BCH102 Lab Manual

BCH 102 Laboratory Manual
M-1

Introductory Biochemistry
Laboratory Manual

BCH 102


M-2
Table of contents

Experiment 1 Purification of native LDH from chicken ........................................................... M-3
Experiment 1A: Buffer and Column preparation ..................................................................... M-5
Experiment 1B: LDH extraction and precipitation ................................................................... M-7
Experiment 1C: Pseudo-affinity chromatography .................................................................. M-11
Experiment 1D: Determining Protein Concentration .............................................................. M-15
Experiment 2 Purification of recombinant lactate-dehydrogenase
from E. Coli by metalion affinity-chromatography .......................................... M-18
(Lab2A-D: Important safety instructions .......................................................................... M-24)
Experiment 2A: Buffer preparation and bacteria inoculation ................................................. M-25
Experiment 2B: Protein Expression ........................................................................................ M-28
Experiment 2C: Purification of (His)LDH by Nickel-bead affinity chromatography ............ M-30
Experiment 2D: Determining Protein Concentration .............................................................. M-33
Experiment 3: Analysis of LDH Purity: SDS-Polyacrylamide Gel
Electrophoresis SDS-PAGE ............................................................................ M-36
Experiment 3B: SDS-PAGE Day 9. Destain SDS gels continued .......................................... M-42
Experiment 4 Yield and Specific Activity of LDH ................................................................. M-43
Experiment 5 K
M
and k
cat
of uninhibited LDH (Kinetics) ....................................................... M-48
Experiment 6 K
M
and k
cat
of inhibited LDH ............................................................................ M-50
Experiment 1-7 - Format for Lab Report ................................................................................ M-52
Experiment 8 Molecular Biology - The transformation, purification
and restriction enzyme analysis of plasmid DNA ............................................. M-57
Experiment 8a: Culturing of E. coli ........................................................................................ M-61
Experiment 8b: Culturing of E. coli ........................................................................................ M-62
Experiment 8c: Plasmid Purification ...................................................................................... M-65
Experiment 9 Restriction analysis of DNA ............................................................................. M-68
Appendix .................................................................................................................................. M-72
Lab Experiment 1 ........................................................................................................... M-72
Lab Experiment 2 ........................................................................................................... M-79
Lab Experiment 5, 6, and 7 ............................................................................................ M-81
References ...................................................................................................................... M-121
Laboratory Report forms Experiments 1-9 ................................................................ M-122


M-3
EXPERIMENT 1-7: Isolation and Characterization of Lactate Dehydrogenase.

(Adapted in 1996 for use in BCH 102 from a series of experiments developed by Professor
Christopher R. Meyer at CSU, Fullerton)

Recommended reading:
Lehninger Principles of Biochemistry, D.L. Nelson and M.M. Cox, editors, 5th edition, Section
6.3, pages 194-205.
This manual, appendices A-D.

INTRODUCTION

Lactate dehydrogenase (LDH, B.C. 1.1.1.27) catalyzes the NAD(H)-dependent
interconversion of lactate and pyruvate:

L-lactate + NAD
+
Pyruvate + NADH + H
+

This enzyme is found in almost all animal tissues, microorganisms, and plants, and plays a key
role in the equilibrium between carbohydrate catabolism and anabolism. For example, the
reduction of pyruvate to lactate (the "reverse" reaction of LDH), which occurs during anaerobic
metabolism, is a key step in regenerating NAD
+
to allow glycolysis to continue. One of the
interesting characteristics of LDH is that it can exist in different "forms" because it is a
tetrameric enzyme with two types of subunits. Depending on the tissue type (i.e., heart muscle
versus skeletal muscle) one type of subunit will be more expressed than the other [H subunit,
"heart"; M subunit, "muscle" (skeletal muscle)]. The tissue-specific differential expression of the
two types of subunits makes the detection and characterization of LDH important in clinical
diagnosis of disease state such as heart disease, where heart muscle damage releases H subunits
of LDH into the blood.
A good method of detection of LDH is to measure its activity - the rate at which substrate is
converted to product under a certain set of conditions. An assay of LDH activity can be
performed by measuring the reduction of NAD
+
to NADH or by measuring the oxidation of
NADH to NAD
+
. These measurements are easily performed because NADH absorbs strongly at
340 nm, while NAD
+
does not. Thus, one can monitor the progress of the reaction by measuring
spectrophotometrically the change in absorbance at 340 nm as a function of time.

GENERAL PLAN OF EXPERIMENT

You will purify the LDH from chicken breast muscle tissue by I) homogenizing the tissue to
release the soluble enzyme, 2) precipitating the enzyme with ammonium sulfate (this is also a
concentration step), 3) absorbing the enzyme to, and subsequently eluting it from, a pseudo
affinity column. This column consists of agarose beads to which dye molecules have been
attached covalently. These dye molecules structurally resemble purine nucleotides. Therefore,
the column absorbs proteins that bind purine nucleotides, allowing them to be separated from
other proteins. The bound LDH can be eluted from the column with NAD
+
or NADH in
moderately high concentrations.

M-4
A fairly rapid purification of LDH can be accomplished by this pseudo-affinity
chromatography technique. Progress of the purification of LDH will be monitored by measuring
both enzyme activity and protein concentration. You will use your purified LDH in subsequent
experiments that involve the kinetic and physical characterization of LDH. However, if your
purification is unsuccessful, then you will be provided with purified LDH for use in the later
experiments.

M-5
Experiment 1: Purification of native LDH from chicken

Experiment 1A: Buffer and Column preparation

METHODS AND PROCEDURES

Day 1. Preliminaries On this day you will prepare three solutions and pour and equilibrate two
columns.

1. Prepare 100 mL of 10 mM Tris-HCl, 1 mM EDTA, pH = 7.4.
Store at 4C. This buffer, after supplementation with a protease inhibitor and a reducing
agent, will be used for extracting LDH from chicken breast muscle tissue.

2. Prepare 500 mL of 10 mM Tris-HCl, pH = 8.6.
Store at Room Temperature. This buffer will be used for packing columns and, after the
addition of a reducing agent, will be used for desalting and column purification of the LDH.

3. Prepare 100 mL of l0 mM Tris-HCl, pH = 8.8.
Store at room temperature. This buffer will be used for LDH enzyme assays.

4. Pour a 10 mL column of Sephadex G-25 in 10 mM Tris-HCl, pH = 8.6.
You will be provided with an empty BioRad Econo-Pak column, a two-way stopcock, and
2.5 g of Sephadex G-25 that has been swollen in 20 mL of 10 mM Tris-HCl, pH = 8.6 in an
18x150 mm test-tube.
a. Securely attach the stopcock to the bottom of the column (it is held in place by
friction).
b. Pour 10 mL of buffer into the column (remember to hold the column at an angle) and
make a mark on the column to note the level.
c. Open the stopcock and let all but a few mL of buffer run out to chase air bubbles from
the frit and stopcock.
d. Gently resuspend the G-25 resin by slowly upending the test tube repeatedly.
e. Pour the resin down the side of the column (remember to hold the column at an
angle).
f. Gently fill the column with some of your 10 mM Tris-HCl, pH = 8.6 buffer.
g. Open the stopcock and let the Sephadex pack. You will need to continually replenish
the buffer above the resin.
h. The column should be completely packed in 10-15 min.
i. If necessary, resuspend the top of the column with a Pasteur pipet and remove resin
so that the total packed volume is 10 mL.
j. Let another 10-20 mL of buffer pass through the column to be sure that it is
completely packed and is 10 mL in volume.
k. Fill the column with buffer.
l. Insert a frit into the column. Open the stopcock and press the frit into the column
barrel, taking care not to trap any air-bubbles beneath it. Using the back end of a 5
mL glass pipet, gently press the frit down the barrel until it just rests on the packed

M-6
resin. This frit will serve as splash guard and will help keep the column from drying
out if you drain the column head.
m. Let 10-20 mL buffer pass through the column after the frit has been positioned.
n. Replace the stopcock with a plug-connector and refill the column with buffer.
o. Cover the top of the column with Parafilm.
p. Store column at 4C.

5. Pour a 3-5 mL column of Cibacron-Blue Agarose in 10 mM Tris-HCl, pH = 8.6.
You will be provided with an empty BioRad Econo-Pak column, a two-way stopcock, and 5
mL of Cibacron-Blue agarose slurry in a small test tube. THIS SLURRY CONTAINS
SODIUM AZIDE - A POISON - AS A PRESERVATIVE. WEAR GLOVES.
a. Securely attach the stopcock to the bottom of the column (it is held in place by
friction).
b. Pour about 20 mL of buffer into the column.
c. Open the stopcock and let all but 5 mL of buffer run out to chase air bubbles from the
frit and stopcock.
d. Gently suspend the resin by slowly upending the test tube repeatedly.
e. Use a Pasteur pipet to transfer the suspended Cibacron Blue resin into the column.
Rinse the test tube with buffer and transfer the rinsings to the column.
f. Gently fill the column with some of your 10 mM Tris-HCl, pH = 8.6 buffer.
g. Open the stopcock and let the resin pack. It should pack to a volume of 3 - 4 mL. You
will need to continually replenish the buffer above the resin.
h. The column should be completely packed in 10-15 min.
i. Let another 10-20 mL of buffer pass through the column to be sure that it is
completely packed.
j. Fill the column with buffer.
k. Insert a frit into the column. Open the stopcock and press the frit into the column
barrel, taking care not to trap any air-bubbles beneath it. Using the back end of a 5
mL glass pipet, gently press the frit down the barrel until it just rests on the packed
resin. This frit will serve as splash guard and will help keep the column from drying
out if you drain the column head.
l. Let 10-20 mL buffer pass through the column after the frit has been positioned.
m. Replace the stopcock with a yellow end-cap and refill the column with buffer.
n. Cover the top of the column with Parafilm.
o. .Store column at 4C.

M-7
EXPERIMENT 1B: LDH extraction and precipitation

DAY 2.

On this day you will take the purification procedure through the ammonium sulfate fractionation
of the homogenized chicken muscle tissue. The enzyme will be stored at 4C as an ammonium
sulfate pellet.

Materials
1. 50 g fresh chicken breast tissue
2. Ice bucket with ice
3. Weigh boat
4. Surgical scissors
5. Six 50 mL centrifuge tubes (for SS-34 rotor)
6. Three screw-capped Eppendorf tubes
7. Graduated cylinder (50 mL)
8. Cheese cloth
9. Beaker (150 mL)
10. Erlenmeyer flash (125 mL)
11. Ammonium sulfate (solid)
12. Magnetic stirrer and stir bar

Procedure.

1. Preparation of 100 mL of EXTRACTION BUFFER [10 mM Tris-HCl, I mM EDTA, 1
mM phenylmethylsulfonyl fluoride (PMSF), 1 mM 2-mercaptoethanol, pH = 7.4].

NOTE: PMSF AND 2-MERCAPTOETHANOL ARE POISONOUS - WEAR
GLOVES.

You prepared 100 mL of 10 mM Tris-HCl, 1 mM EDTA, pH = 7.4 on day 1.

To this solution add PMSF to a final concentration of 1 mM and 2-mercaptoethanol to a final
concentration of 1 mM. The stock solution of PMSF is in the hood (0.10 M PMSF in
absolute ethanol). The beta-mercaptoethanol is also in the hood (pure beta-mercaptoethanol
is 14.3 M). Before pipeting the PMSF or beta-mercaptoethanol, have the TA's check your
calculations:

Volume of 0.10 M PMSF needed: ____________

Volume of beta-mercaptoethanol needed: ____________

2. Preparation of tissue.
a. Remove any skin or large clumps of fat from the chicken breast tissue.

M-8
b. Mince the tissue into fine pieces (about Y2 the size of a thumb nail) with the surgical
scissors, placing the pieces into the chilled weigh boat.

3. Preparation of glassware, centrifuge tubes, and Eppendorf tubes (prepare this during
the centrifugation)
a. Place the stir-bar in the 150 mL beaker, cover the beaker with Parafilm, and place the
beaker in the ice-bucket so that the stir-bar is centered on the magnetic stirrer. Pack ice
around the beaker. (The Parafilm will prevent ice from falling into the beaker. When you
have finished adding the ice, you can remove the Parafilm).
b. Pour 35 mL of water into two centrifuge tubes. Mark the levels with ink, empty the
tubes, and store the tubes on ice.
c. Place the four other 50 mL centrifuge tubes and three Eppendorf tubes on ice.
d. Place your large weigh boat on ice.
e. Label your three screw-capped Eppendorf tubes "crude extract" and label them with your
section and station number. Use tape. Also write your station number on the cap. Place
the tubes on ice.

4. Homogenization.
a. Remove a blender/blade assembly from the cold box. Place your minced chicken tissue
into the bottom of the blender and carefully pour your 100 mL of extraction buffer onto
the chicken (remember that the buffer contains the poisons, PMSF and 2-
mercaptoethanol).
b. Securely place the top of blender/blade assembly into position. BE SURE THAT THE
TOP IS ON CORRECTLY - OTHERWISE YOU WILL SPRAY YOURSELVES AND
OTHERS WITH PMSF AND beta-MERCAPTOETHANOL WHEN YOU TURN ON
THE BLENDER!
c. Carry the capped blenderlblade assembly to a motor housing. Homogenize with four
bursts of 30 s each, waiting 30 s between bursts to permit the heat generated by the blades
to dissipate.
d. Pour 35 mL of homogenate into two chilled, marked, centrifuge tubes.
e. Use a balance to adjust the contents of the two tubes so that their weights are the same to
within 0.1 g.

INITIAL WEIGHTS: Tube 1: __________ Tube 2: __________

FINAL WEIGHTS: Tube 1: __________ Tube 2: __________

f. Centrifuge the tubes for 20 min at 15,000 rpm (see TA for help with the centrifuge).
When centrifugation is finished and the rotor has stopped, you should see large pink
pellets, flesh-colored supernatents, with a floating layer of white fat.
g. Pour both supematents through two layers of cheesecloth into the chilled 150 mL beaker.
The floating layer of fat should remain trapped in the cheesecloth.
h. Place 0.5 mL aliquots in each of the three chilled Eppendorf tubes. Keep these tubes on
ice. They will be flash-frozen in liquid nitrogen and stored in the -20C freezer.
i. Measure the volume of the remaining supernatant with the graduated cylinder.

M-9
5. Ammonium sulfate fractionation/precipitation (40% cut).
a. Weigh out 0.24 g of solid ammonium sulfate for each 1 mL of supernatant that you have.
b. Break up any ammonium sulfate chucks with a spatula
c. Slowly (over 15 minutes), with stirring, add the solid ammonium sulfate to the
supernatant. When you have finished, the concentration of ammonium sulfate will be
40% of saturation.
d. Let the solution stir for an additional 15 min to allow the ammonium sulfate/protein
mixture to reach equilibrium.
e. Distribute the ammonium sulfate solution between the two remaining, chilled, centrifuge
tubes. Balance to within 0.1 g:



f. Centrifuge the tubes for 20 min at 15~000 rpm (see TA for help with the centrifuge).
When centrifugation is finished and the rotor has stopped, you should see small
fleshy/pink pellets beneath a clear light-pink supernatant.
g. Pour both supernatants into a chilled 125 mL glass beaker. These supernatants are 40%
saturated in ammonium sulfate and at this point contain most of the LDH.
h. Measure the volume of the 40% saturated supernatants by using a graduate cylinder. Pour
the supernatant back into the chilled 125 ml glass beaker. Keep on ice.
i. Wrap the centrifuge tubes containing the 40% pellets with Parafilm and place these in a
rack in the 4C refrigerator (wait for instructions) (you will keep these in case something
went wrong and your enzyme is in the pellet!).

6. Ammonium sulfate fractionation/precipitation (60% cut).
a. Weigh out 0.13 g of solid ammonium sulfate for each 1 mL of 40% saturated ammonium
sulfate supernatant that you have obtained in step 5. Break up any ammonium sulfate
chucks with a spatula.
b. Slowly (over 15 minutes), with stirring, add the additional solid ammonium sulfate to the
40% supernatant. When you have finished, the concentration of ammonium sulfate will
be 60% of saturation.
c. Let the solution stir for an additional 15 min to allow the ammonium sulfate/protein
mixture to reach equilibrium.
d. Distribute the ammonium sulfate solution between the two remaining, chilled, centrifuge
tubes. Balance to within 0.1 g:



e. Centrifuge the tubes for 20 min at 15,000 rpm (see TA for help with the centrifuge).
f. When centrifugation is finished and the rotor has stopped, you should see again small
fleshy/pink pellets beneath a clear light-pink supernatant.

M-10
g. Pour the supematents into a 125 mL Erlenmeyer flash, cover with Parafilm, and store at
4C (in case something went wrong and the enzyme didn't precipitate).
h. Wrap the pellet-containing centrifuge tubes with Parafilm (these pellets should contain
most of the enzyme!) and place these in a rack at 4C.


M-11
Experiment 1C: Pseudo-affinity chromatography

Day 3.

On this day you will: 1) resuspend and desalt your enzyme solution; 2) bind the LDH to the
Cibacron blue agarose column; and 3) elute the enzyme it with NADH. You will monitor the
progress of protein elution from the agarose column by monitoring the absorbance of the
fractions at 280 nm.

Materials.

For column purification:
1. Sephadex G-25 column (poured on day 1).
2. Cibacron Blue agarose column (poured on day 1)
3. 30 test tubes (13 x 100 mm) for collecting fractions.
4. Quartz cuvette
5. 10 mL of NAD
+
buffer [10 mM Tris-HCL, pH = 8.6, 0.5 mM 2-mercaptoethanol, 1 mM
lithium lactate, 1 mM NAD
+
.
6. 40 mL of NADH buffer [10 mM Tris-HCl, pH = 8.6, 0.5 mM 2 mercaptoethanol, 1 mM
NADH.
7. Three screw-capped Eppendorf tubes.

Procedure.

1. Preparation of 200 mL of TRIS-ME BUFFER (10 mM Tris-HCl, 0.5 mM 2-
mercaptoethanol, pH = 8.6).

RECALL THAT 2-MERCAPTOETHANOL IS POISONOUS - WEAR GLOVES.

You prepared 500 mL of 10 mM Tris-HCl, pH = 8.6 on day 1. To 200 mL of this solution,
add 2-mercaptoethanol to a final concentration of 0.5 mM. The 2-mercaptoethanol is in the
hood (pure 2-mercaptoethanol is 14.3 M). Before pipetting the 2-mercaptoethanol, have the
TA's check your calculations:

Volume of 2-mercaptoethanol needed: __________

M-12
2. Chill some TRIS-ME buffer and the Eppendorf tubes.

a. Place 2 mL of TRIS-ME buffer in a test tube on ice.
b. Label the 3 Eppendorf tubes "AmS0
4
" and label them with your section and station
number. Use tape. Write your station number on the cap. Place the tubes on ice.

3. Preparation of columns. Both columns should be at room temperature. They will have been
removed from the cold room during the morning before the lab session. Recall that both
columns were poured in 10 mM Tris-HCl, pH = 8.6. It is now necessary to equilibrate them
with buffer containing 0.5 mM 2-mercaptoethanol.
a. Remove the Parafilm covers from the columns and replace the yellow end-caps with two-
way stopcocks.
b. Drain both column heads down to the upper frits.
c. Fill both columns with TRIS-ME buffer and drain until only 3-4 mL of buffer remains
above the packed resin.
d. Close both stopcocks.

4. Suspension of Ammonium Sulfate Pellet.
a. Resuspend the pellet in a minimal volume of TRIS-ME buffer. To do this, add I mL of
TRIS-ME buffer to one pellet and gently suspend the pellet using the squeeze-bulb end of
a plastic Pasteur pipet by gently rubbing the bulb up and down against the pellet. Avoid
the creation of bubbles/foam. Protein/enzymes denature at the surface of these bubbles
because of surface tension interactions.
b. After the first pellet is suspended (the total volume should be just over 2 mL), transfer the
suspension onto the second pellet and suspend that pellet in the same manner as the first.
The total volume of the two-pellet suspension should be just over 3 mL. The suspension
will be pale yellow and slightly cloudy. Keep the suspension on ice.

5. Desalting with Sephadex G-25 column. The 10 mL G-25 column can desalt 3 mL of
sample into 4 mL.
a. Drain remaining buffer in column down to level of frit and close stopcock.
b. Place 3.0 mL of resuspended sample onto column.
c. Open stopcock and let column drain to frit. Set aside the 3 mL flowthrough (on ice).
Close stopcock.
d. Place 4 mL TRIS-ME buffer onto column.
e. Open stopcock and collect 4 mL flowthrough. Store on ice. This fraction contains your
desalted enzyme/protein solution. Cover this tube with Parafilm and upend it gently a few
times to make sure it is homogeneous.
f. Fill column with TRIS-ME buffer and collect 4 mL fractions until cloudy material elutes
(containing lipid/salt). Let buffer drain to frit.
g. To clean the column, let the TRIS-ME buffer drain to the frit, fill the column with 10 mM
Tris-HCl, pH = 8.6 buffer prepared on Day 1, and let column drain to just above frit.
Close stopcock.
h. Place 100 L aliquots of your desalted enzyme/protein solution into each of the three
chilled Eppendorf tubes. Keep these tubes on ice. They will be flash-frozen in liquid
nitrogen and stored in the -20C in the freezer.

M-13
i. Measure the volume of your remaining desalted enzyme/protein solution.

6. Cibacron Blue Chromatography (collect 5 mL fractions and label them).

Loading the column.
a. Open the stopcock and let the buffer drain to the frit.
b. Load your approximately 4 mL of desalted enzyme/protein fraction onto the column.
(Record the exact volume of the fraction that you load).
c. Open the stopcock and begin collecting the first 5 mL fraction.
d. Rinse the inner wall of the column with a few mL of TRIS-ME buffer and let the rinsings
drain to the frit.

First Wash.
e. Fill column with TRIS-ME buffer.
f. Collect 5 mL fractions, label, and store on ice.
g. Read the A
280
of the fractions (use TRIS-ME buffer as the blank). Continue collecting
fractions until the A
280
is less than 0.1. (Five or six fractions should be sufficient.) Add
TRIS-ME buffer to top of column as needed. What component(s) absorb at 280 nm?

Second Wash
h. Let buffer in the column drain to the frit.
i. Add 10 mL of NAD
+
(Do not use NADH this will elute your protein!!!) buffer to column.
(This should elute weakly-bound dehydrogenases.)
j. Collect 5 mL fractions, label, store on ice, and read their A
280's
. What component(s)
absorb at 280 nm?

Third Wash
k. Fill column with TRIS-ME buffer.
l. Collect 5 mL fractions. Label them. Store them on ice. Read their A
280's
. Continue
collecting fractions until the A
280
of the eluted solution is less than 0.1. (Five fractions
should be sufficient). Add TRIS-ME buffer to top of column as needed.

Elution
m. Let buffer in column drain to frit.
n. Fill column with NADH buffer that can be obtained from the front of the room at this
time. (This should elute the LDH).
o. Collect 5 mL fractions; label them; store them on ice; and read their A
280's
. Collect 6
fractions. Add NADH buffer to the top of the column as needed. What component(s)
absorb at 280 nm?
p. To wash the column, let buffer drain to the frit; fill the column with 10 mM Tris-HCl, pH
= 8.6, prepared on day 1; and let column drain until only a few mL of buffer remains in
column.

7. Prepare a graph showing the A
280
as a function of fraction number for the fractions that
eluted from the Cibacron Blue column with NADH.


M-14
8. Keep all fractions on ice.
9. Storage of your Cibacron Blue fractions.
a. Place Fractions 2 - 6 at 4C.
b. Cover Fraction l (one) with Parafilm and upend it gently a few times to make sure it is
mixed homogeneously. Aliquot this fraction into five CHILLED AND LABELED 1 mL
Eppendorf tubes. These aliquots will be frozen in liquid nitrogen by your TA's and
stored at -20C.
c. The 100 L aliquots of your desalted ammonium sulfate fraction will also be frozen in
liquid nitrogen with the help of your TA's and stored at -20C.


M-15
Experiment 1D: Determining Protein Concentration

On this day, you will determine the protein concentration, in mg/mL, of your crude extract, your
desalted ammonium sulfate fractions, and your pseudo-affinity purified LDH fractions.

Materials.
For Bradford Assays:
1. Concentrated Bradford reagent (Bio-Rad).
2. BSA at 0.2 mg/mL in 10 mM Tris-HCl, pH = 8.6.

To determine the yield of LDH, and so that you can determine the specific activity of the enzyme
at various steps in the purification procedure, you must determine the concentration of protein in
your: 1) crude tissue homogenate (thaw one of your 0.5 mL aliquots); 2) desalted ammonium
sulfate fraction (one of your 100 L aliquots); and 3) the first four fractions that were eluted
from your Cibacron Blue column by NADH.

Table I. Standard Curve table
Assay # Protein
Added
(g)
200
ug/mL
BSA
Stock
(l)
Sample
Type
10 mM
Tris-
HCl, pH
= 8.6
Bradford
Reagent
(l)
Final
Volume
(mL)
A
595
Measured
Protein
(g)
1 0 0 Blank 800 200 1.0
2 2 BSA 200 1.0
3 4 BSA 200 1.0
4 6 BSA 200 1.0
5 8 BSA 200 1.0
6 10 BSA 200 1.0


M-16

Table II. Sample preparation table
Assay # Sample
dilution
factor
Volume
added
(l)
Sample
Type
10
mM
Tris-
HCl,
pH =
8.6
Bradford
Reagent
(l)
Final
Volume
(mL)
A
595
Measured
Protein
(g)
1 100 10 Crude 200 1.0
2 100 50 Crude 200 1.0
3 100 10 Desalted 200 1.0
4 100 50 Desalted 200 1.0
5 1 10 NADH1 200 1.0
6 10 10 NADH1 200 1.0
7 1 10 NADH2 200 1.0
8 10 10 NADH2 200 1.0
9 1 10 NADH3 200 1.0
10 10 10 NADH3 200 1.0
11 1 10 NADH4 200 1.0
12 10 10 NADH4 200 1.0

Note: All Samples described in Tables 1 and 2 have to set-up at the same time. Do not measure
the BSA standard without the protein samples and vice versa. The BSA standard is only useful
when coupled to the protein samples.

1. You will set up tubes for the Bradford assay The BSA standards and your LDH fractions
should be diluted with 10 mM Tris-HCl, pH = 8.6. For your standard curve, use 0, 2.0, 4.0,
6.0, 8.0, and 10.0 g of BSA in a total volume of 0.8 mL.
REMEMBER THAT YOUR LDH FRACTIONS CONTAIN TRACES OF THE
POISON, 2-MERCAPTOETHANOL. WEAR GLOVES.
2. Set up tubes for your LDH fractions (use 10 mM Tris-HCl, pH = 8.6, as the diluent and be
sure that each tube contains a total volume of 0.8 mL). These fractions will have to be
diluted so that their A
595
values will fall in the range of the BSA standards. For the Crude
homogenate and the desalted ammonium sulfate fractions, try using 10 L and 50 L of a
100-fold dilution (diluted into 10 mM Tris-HCl, pH = 8.6). For your four Cibacron Blue
NADH and Ni-Affinity fractions, try using 10 L of the undiluted fraction and 10 L of a
10-fold dilution (diluted into 10 mM Tris-HCl, pH = 8.6).
PREPARE THE APPROPRIATE TABLE INCLUDING CALCULATIONS FOR
YOUR DILUTIONS IN YOUR NOTEBOOK BEFORE COMING TO THE
LABORATORY.
3. To start the Bradford assay, you will need to add 200 L of concentrated Bradford
reagent to each tube. Do so only after all samples have been prepared and assay can be
performed on all samples within the same amount of incubation time. Mix thoroughly

M-17
and let each tube incubate for at least 5 min (but no longer than 50 min) at room
temperature. Record the A
595
value of each tube.
4. Prepare a graph that shows the A
280
(determined on day 3; plot protein concentration and
A280 on the same graph) and the concentration of protein (in mg/mL) as a function of
fraction number.
5. Complete purification table. Using the obtained protein concentrations calculate Yield of
purification as a means to evaluate the success of the purification procedure

Fraction
Volume
(mL)

Concentration
of Protein
(mg/mL)
Purification (%)
(based on protein
concentration)
Crude Extract

Desalted
Ammonium
Sulfate Fraction

Peak NADH
fraction from
Cibacron Blue
column

Peak
Recombinant
LDH fraction


M-18
Experiment 2: Purification of recombinant lactate-dehydrogenase from E. Coli by metalion
affinity-chromatography
Background
The ability to express proteins in bacteria has made possible many structural and functional
studies that were not previously possible due to limitations in protein availability. It has also
proven invaluable in producing proteins for drugs, immunizations and antibody production. This
has been especially true for low abundance proteins, such as transcription and translation factors,
hormone receptors, signal transduction proteins, and cell-cycle regulatory proteins. By cloning
genes for these proteins, inserting the genes into plasmids, and transfecting the plasmids into
bacteria, milligram quantities of the target protein can be obtained from less than a liter of cells.
Such systems are termed overexpression systems, because the target protein is transcribed from
a very strong promoter and can account for 10% or more of the total cell protein.
In this experiment you will isolate a recombinant His-tagged, Lactate-Dehydrogenase
[(His)LDH] from E. coli by metalion affinity-chromatography. The gene for the LDH was
isolated from the fly Drosophila melanogaster. Similar to chicken LDH, fly LDH is active as a
tetramer and each subunit has a mass of 38,000 Daltons.

Expression system:
In choosing a cloning system for a specific protein, several things must be considered. One
important consideration is whether the protein is toxic to the host bacterial cell. High
transcription rates can lead to slow cell growth, due in part to the metabolic demands of
translating high levels of the foreign protein. If gene product diminishes the host cells growth
rate, the foreign protein is considered toxic.
Recombinant proteins with exposed hydrophobic regions on their surface also often have toxic
effects on host cells, probably because of their incorporation into vital membrane systems.
Membrane proteins, proteins that interfere with electron transport, and many DNA binding
proteins, such as the transcription factor you will isolate are toxic. Even though the DNA binding
proteins recognize very specific DNA sequences, when they are over-expressed to high
concentrations they will bind non-specifically to the host chromosome, interfering with its
activity. It is impossible to maintain a cell line with a toxic gene insert, if that gene is expressed.
To circumvent this problem, most modern expression systems utilize an inducible viral
promoter for the target gene, which is not recognized by the host cell. The target gene is not
expressed in the host cells until an inducer is added to the culture. Only after the cells have
reached a high density is the target gene induced, and the cells are harvested a few hours later.
The LDH gene was cloned into a plasmid (pet19b, engineered by Novagen; see Figure 1). The
expression of the LDH is controlled by a bacteriophage T7 promoter (Figure 2). This requires T7
RNA polymerase for expression. The insertion of the cDNA for LDH into pet19b creates a
fusion cDNA, which expresses a fusion protein that consists of LDH and a few specific amino
acids, which include a series of histidine amino-acids (His-tag), at the NH
2
-terminus of LDH.
The resulting recombinant LDH is slightly longer than the native protein, and contains a series of
6 histidine residues at the NH
2
-terminus. This His-tag bind to nickel (they form a coordination
complex). A nickel (metal) affinity affinity-chromatography allows for a rapid, efficient one-
step affinity purification of the recombinant (His)LDH.

M-19

Figure 1. pet19 plasmid map

M-20

Figure 2. Plasmid map of [pet19b(His)LDH] . The location of LDH cDNA is labeled.

Several options are available for expression of a protein whose complementary DNA sequence is
known. Sequences encoding a minimum of six histidines (i.e., CATCATCAT) must be added
to either the amino or carboxyl terminus end of the coding portion of the gene. There must be an
initiator methionine at the amino terminus and a termination codon at the carboxyl terminus. To
do this easily and quickly the cDNA can be subcloned into a vector, which already contains the
deca-histidine sequence with compatible restriction enzyme sites adjacent to the histidine
sequences. Qiagen, Novagen, and Invitrogen, among others, sell these expression systems,
which also contain protease cleavage sites (e.g.. thrombin) adjacent to the oligo-His sequences to
allow removal of the His tail after purification of the overexpressed protein.
The His-tag can be inserted at the amino- or carboxyl-end of a protein. For very long
recombinant proteins that may be subject to premature termination, placing the tag at the
carboxyl-terminus will select only for full-length proteins during purification. Proteins, which
must be secreted for proper folding or processing (or to remove highly toxic proteins from the
cells), contain a signal sequence for this in the amino-terminal region. Here, the His tag must be
on the carboxyl-end so it will not to interfere with or be cleaved during processing. On the other
hand, initiation of translation at internal start sites can occur if the initial ribosome consensus
sequence is not a strong enough Kozak sequence. Placement of the His tag sequence at the
amino-terminal of the protein will prevent the co-purification of shorter proteins whose synthesis
starts at internal AUG sites (Fig. 3).


M-21

Figure 3. Map and amino-terminal 10xHis tag Construct pet19b

Removal of the His-tail from the protein is not always necessary. 1) These tails do not interfere
with the structure or function of most proteins, including enzymes, transcription factors, and
proteins used to make vaccines. 2) The His tag is uncharged at physiological pH, it is small and it
does not generally interfere with function, folding, compartmentalization, or secretion. 3) The
His tail is poorly immunogenic, and thus the recombinant protein can be used directly without
removal of the tag as an antigen to generate antibodies against the protein. Further advantages of
the tag are: 1) the protein may be immobilized on metal-chelating surfaces which can simplify
protein interaction studies, and 2) anti-His antibodies are available commercially, and can be
utilized for identification of the target protein or other proteins it interacts with. A His-tag plus
the small protein dihydrofolate reductase (6xHisDHFR) tag has also been utilized in some
circumstances. For example, if the protein of interest is very small (<10 kDa), it may be
degraded very rapidly in E. coil. If the short protein is expressed as a fusion with DHFR it is less
likely to be degraded. Since the His-tag tag is not highly immunogenic in mouse and rat, peptides
fused to the tag can be used directly for immunizations or epitope mapping. If degradation of the
His-tagged protein is still a problem, one can also reduce the growth temperature, induce the
protein for shorter time periods, use a host strain deficient in one or more proteases, or, add low
concentrations of protease inhibitors such as PMSF (<1 mM) in the culture medium before
induction. Note that PMSF is toxic to humans.
The (His)LDH encoding pet 19b plasmid [pet19b(His)LDH] will be used in our studies. We
have transfected the pet19b(His)LDH plasmid into an E. coil cell strain derived from the BL21
E. coli strain. These cells have been engineered by Novagen to contain a lysogen of
bacteriophage DE3 (i.e., a virus has been integrated into the host cell chromosome). DE3
contains the gene for T7 RNA polymerase, under an IPTG (isopropyl- -D-
thiogalactopyranoside) inducible lactose (lac) promoter. The addition of 0.1-1 mM IPTG to the
bacteria culture induces synthesis of T7 RNA polymerase, which initiates expression of
(His)LDH from the T7 promoter present in the pet19b. These cells have an additional feature,
pLysE, a plasmid containing the T7 lysozyme gene. T7 lysozyme inhibits T7 RNA polymerase,
ensuring that no (His)LDH is made due to leaky polymerase activity. When IPTG is added, so
much polymerase is made that lysozyme cannot inhibit it, and LDH is expressed. Expression of
lysozyme in the cells also greatly facilitates preparation of cell extracts. E. coli tolerates fairly
high levels of lysozyme, because the protein is unable to pass through the inner membrane to

M-22
reach the peptidoglycan cell wall. Treatments that disrupt the inner membrane, but do not
normally cause cell lysis, induce rapid lysis of cells overexpressing lysozyme.
Purification by metal-ion affinity chromatography
The metal-affinity matrix that will bind proteins with a series of histidine residues is composed
of a solid support (Sepharose CL-6B beads), covalently bound to a short carbohydrate chain
(spacer) ending in a nitrilotriacetic acid (NTA) functional group. NTA has four chelating sites to
coordinate a Ni
2+
ion, leaving two Ni
2+
coordination sites free to bind to two adjacent histidine
residues.

When the resin is charged with nickel ions, it has a light blue-green color, and when stripped of
nickel, it is white. NTA-metal binding is more stable than other chelating agents and retains the
ions under a wide variety of stringent wash conditions. When cell lysates are passed through Ni-
NTA columns, the His-tagged proteins bind selectively to the Ni
2+
. The column is then washed
and the protein eluted with imidazole, which competes with the protein for the Ni binding sites
(the imidazole ring looks much like histidine). Low levels of imidazole are included in the lysis
and wash buffers to minimize non-stringent interactions of other proteins with the resin.

At low imidazole concentrations, the affinity of the ten-histidine residues in the 10xHis tag is
strong, allowing efficient binding of the 10xHis-tagged proteins, whereas non-specific binding of
background proteins is prevented. Usually up to 20 mM imidazole can be used, although if the
tagged-protein does not bind, the concentration must be reduced. This results in quite a clean
protein preparation, although some E. coli metal binding proteins may also bind to the column
and elute with (His)LDH. Using this method, often a protein which comprises <1% of the total
cellular protein can be purified to more than 95% homogeneity in just one step.

Figure 4 Interaction of Ni-NTA matrix with histidine residues

Binding of tagged proteins to the Ni-NTA resin is not dependent on the protein conformation.
Non-ionic detergents, denaturing agents (e.g., urea, guanidine HCl), and relatively high salt
Two His residues of His-tag attached to a complexed Nickel
ion in Ni-NTA
Ni-NTA
spacer
Matrix
(resin)

M-23
concentrations (2 M NaCl) do not interfere. This can be of importance in purification if
nonspecific binding to the matrix occurs due to nonspecific hydrophobic or ionic interactions.
Additionally, DNA or RNA bound to proteins may also be removed. Since it is not dependent on
tertiary structure, proteins can be purified in the native or denatured state. The decision to purify
a protein in the denatured or nondenatured state depends, in part, on how they are expressed in
the cells (i.e., if they are insoluble in inclusion bodies, they may have to be denatured to
solubilized them.) Also, in rare circumstances the 10xHis-tag is buried in the molecule, and
under these conditions, denaturing conditions would have to be utilized. Proteins that require
strong reducing agents for stability, like -mercaptoethanol at >10 mM, cannot be purified with
this procedure since the nickel will be reduced and cannot bind histidines. The column would
turn brown. If proteins are purified under denaturing conditions, there has to be a method for
renaturation of the protein after it is purified. This is most often done by long dialysis to remove
denaturants and allow for refolding. However, this will not work for many proteins.
When cells are lysed, several steps can be taken to optimize the purification procedure. 1) To
prevent degradation, cells and protein should be kept at 0-4 C at all times. Protease inhibitors
may also need to be added. 2) Imidazole at low concentrations can help minimize nonspecific
protein binding. 3) Relatively high ionic strength in the buffers (i.e., addition of 300 mM-2 M
NaCl in the buffers) will also help prevent nonspecific interactions between the protein and the
matrix.
The His-tagged protein can be purified by a batch or column procedure. In the batch procedure,
the protein is mixed with the matrix followed by pouring the mixture into a column and
proceeding with washing and eluting. This has advantages over adding the protein sample to a
prepoured column of resin if the protein is in the cell lysate in very low concentrations, or the
accessibility of the 6xHis tag is limited. Washing and eluting buffers also must be tested to
determine the pH, ionic strength, and imidazole concentrations that are optimal for reducing
contaminating proteins and producing maximum protein recovery. The histidine residues have a
pKa of ~6.0 and will become protonated if the pH is reduced (pH 4.5-5.3). Monomeric proteins
will generally elute at about pH 5.9, whereas dimers and aggregates having more than one
10xHis-tag will elute closer to pH 4.5. If this occurs the 6xHis-tagged protein can no longer bind
to the nickel ions and will elute from the column. Similarly, if the imidazole concentrations are
raised above 100 mM, the protein can no longer compete and it will be eluted. A third method
utilizes chelating agents such as EDTA. These agents will complex with the nickel in the column
and strip it from the NTA groups. The 10xHis-tagged protein will elute as a nickel-protein
complex that may or may not be desired. The column will turn white since the nickel has been
removed, and would have to be recharged before being reused. From this, one can see that there
are a variety of approaches; changing the imidazole concentration tends to be the mildest
procedure, since is it least damaging to the structure of the protein. For each protein, the
particular reproducible elution conditions must be determined empirically.

M-24
Lab2A-D: Important safety instructions
- All materials, which come into contact with transfected cells, have to be autoclaved prior to
disposal, so all plastic ware, which came in contact with live bacteria, should be disposed of in
the plastic biohazard bag. The bacteria culture supernatant will be saved in a designated
biosafety liquid waste container.
- Because we will be working with recombinant bacteria, the experiments are defined as
biosafety level 1 experiments. A lab coat is required. You cannot perform the experiments in
Lab2A and Lab2B without lab coat.
- On day three, you will be working in a 4C cold room (This is around 38 Fahrenheit). Come
prepared. We recommend winter jacket, scarf, warm hat, finger gloves, and winter shoes.

M-25
Experiment 2A: Buffer preparation and bacteria inoculation
DAY 4.

Materials Provided

Tris- 2g (M 121.14 g/mol)
HCL 1M- 5 mL
NaOH 1M- 5 mL
Glycerol (20% stock solution) 20 mL
Triton-X 100 2%- 2 mL
1M Imidazol- 40 mL
NaCl- 6g (M: 58.44 g/mol)
LB Broth Powder- 7g
30 mL bacteria culture tube with lid- 1
5 mL Serological Pipette- 1
5 and 25 mL glass pipette
3 x 50 mL glass beaker
3 x glass bottles (or 50 mL Falcon tubes)
3 x 50 mL graduate cylinder
500 mL Erlenmeyer flask
Aluminum foil
Biohazard waste bag


M-26
I. Buffers
Prepare lysis, wash, and elution buffer. Store all buffers at 4C before you leave. Label bottles
with station number, section number, and names.

a. 50 mL Lysis Buffer (Lysis)
50 mM Tris pH 7.9
500 mM NaCl
2% glycerol
0.02% Triton-X100
1 mM PMSF (toxic, carcinogen). Note: PMSF will added shortly before you are ready to use the
buffer on day 2.
5 mM Imidazole

b. 50 mL Wash Buffer (Wash)
50 mM Tris pH 7.9
500 mM NaCl
2% glycerol
0.02% Triton-X100
buffer on day 3.
60 mM Imidazole

c. 50 mL Elution Buffer
50 mM Tris pH 7.9
500 mM NaCl
2% glycerol
0.02% Triton-X100
buffer on day 3.
500 mM Imidazol

Store all three buffers at 4C.

M-27
II. Bacteria Media

LB Medium
Prepare 250 mL Lucia Broth Medium (LB medium): Dissolve 6.25g LB Broth, Miller in 250 mL
ddH
2
O. Once dissolved fill medium into 500 mL Erlenmeyer flask. Seal flask with aluminum
foil. Place autoclave tape on flask. Label tape with your name, section number, and station
number. Once completed, the TAs and lab coordinator will assist you with autoclaving the
prepared media.

III. Inoculate bacteria 5 mL LB culture
You need to inoculate the (His)LDH-expressing bacteria on this day. Remove 5 mL LB medium
containing 200 g/mL ampicillin from the provided stock. Place the 5 mL in the provided sterile
30 mL bacteria culture tube. Label glass tube with your name, section number, and station
number. By using a sterile pipette tip, remove one colony of E. coli Bl21-(His)LDH from the
provided plates. Drop pipette tip in the 5 mL medium. Once completed, place bacterial culture in
37C incubator.

M-28
Experiment 2B: Protein Expression

Day 5.

Materials Provided

15 mL Plastic tube- 4
Plastic cuvette- 1
Biohazard waste bag- 1
5 mL Serological or Sterile Pipette tip- 3
1 M IPTG- 200 l
100 mM PMSF
Centrifuge bottle- 1

1. Retrieve your 200 mL bacterial culture from the 37C incubator. (Note: This is your LB from
Day 1 with your culture added to it several hours before class, in order to allow the culture to
reach an appropriate OD
600
in a timely manor)

2. Remove 4 mL of the bacterial culture, place the 4 mL into a 15 mL plastic tube, and store the
bacteria on ice on ice (see step 4).
3. Measure absorption at fixed wavelength 600 nm (OD
600
) (You will use the instructions found
on the old Beckman spectrophotometers in the 102 Lab; place your cuvette in the right
orientation; if you have never done this before, ask for advise before using the
spectrophotometer).

If the OD
600
of your culture is <0.3, wait till the culture reaches a OD
600
of 0.3. Please keep in
mind that bacteria will double every 15-20 minutes.
If your culture has reached an OD
600
>0.3 your culture is ready. Induce expression of the
recombinant LDH by adding 1mM IPTG (final concentration). We shall provide you with a 1 M
IPTG stock solution. You have to calculate the volume of IPTG stock solution necessary to
induce expression before you come to the lab.
After induction, put the culture back into the 37C incubator. The total incubation time for
adequate protein expression is 3h.

4. Transfer 1.5 mL of the saved bacteria sample into a 1.5 mL reaction tube. Collect the bacteria
by centrifugation (5 min, room temperature, 13k RPM).
- Discard the supernatant.

M-29
- Resuspend bacteria in 75 L H
2
O and add 25 L 4 x protein sample buffer. Mix well. Boil
samples 5 min in 100C heat block.
- Place sample on ice.
- After 5 min, save sample in -20C freezer.
- Label tube with your section and station number and the word uninduced

5. After 1h and 2h remove 4 mL of the bacterial culture, transfer into a 15 mL plastic tube, and
store the bacteria on ice. Measure and record OD
600
.
For each time point, process 1.5 mL of the saved bacteria as described in step 4. Save the lysates
at -20C. Label the tubes with your station and section number and the symbols 1h and
2h, respectively.

6. After 3h remove 4 mL of the bacterial culture, transfer into a 15 mL plastic tube, and store the
bacteria on ice. Process 1.5 mL of the saved bacteria as described in step 4. Label tube with
section and station number and the word induced).
Measure and record OD
600
.

7. Collect the bacteria of the 200 mL culture by centrifugation (10 min, 5000 RPM, 4C).
While your sample is in the centrifuge retrieve your lysis buffer from the refrigerator, add PMSF
(1 mM final; Calculate the required volume based on a 100 mM PMSF stock solution).

8. Carefully discard all supernatant without losing any of the bacterial pellet. Place centrifuge
tube on ice. Resuspend bacteria pellet in 5 mL lysis buffer by using a sterile 10 mL pipette.
Transfer the homogenate into a 15 mL plastic tube. Flash freeze the homogenate (the TAs and
laboratory coordinator will assist you). Store the frozen bacteria at -20C (wait for instructions).
Place lysis buffer back into the refrigerator, you will need it again for Lab 2C.


M-30
Experiment 2C: Purification of (His)LDH by Nickel-bead affinity chromatography

DAY 6.

Materials Provided

50 mL beaker- 1
Column assembly (Column, Stopcock, Yellow cap, Column stand)- 1
Screw cap tubes- 15
100 mM PMSF- 200 l
15 mL Plastic tube- 1
30 mL centrifuge tube with lid- 1
Nickel Affinity beads 50% suspension- 1 mL
DNase
Lysozyme
100 mM PMSF

Important notes:
- Keep your protein sample on ice all times.
- Come prepared, otherwise this will become a very long day
- Bring your cold room clothes: without these you cannot perform the
experiment.

1. Defrost your bacterial homogenate on ice. Retrieve your lysis buffer from the refrigerator.
Replenish the PMSF. Place 50 mL beaker on ice. Put magnetic stir bar into the beaker. Place
your magnetic stir plate, 15 screw cap tubes, 1.5 mL reaction tubes, plastic rack for 1.5 mL
reaction tubes, and column equipment into the 4C room.

2. Add 5 mL of lysis buffer to the bacterial homogenate.

3. Once completely thawed, transfer the 10 mL homogenate into the cold 50 mL beaker. Add 0.5
mL lysozyme stock solution (stock: 100 g/mL) and 0.5 mL DNase stock solution (stock: 50
g/mL).
Place beaker on stir plate in the cold room. Stir the solution at low speed. Incubate for 30 min.

4. Transfer solution into 15 mL plastic tube. Sonicate the sample (the laboratory coordinator and
TAs will assist you with this step).

5. Transfer homogenate into a 30 mL centrifuge tube. Centrifuge sample at 15 min, 15.000 RPM
at 4C.

M-31

Steps 7-12 will be performed in the cold room. Please do not forget to bring appropriate clothing
(warm jacket, gloves, hat)

6. During the centrifugation run, prepare the Ni-affinity agarose (Ni-beads).
Wash the Ni-beads with lysis buffer. Add 10 ml lysis buffer to the 15 ml falcon tube, which
contains the Ni-beads. Mix well for 2 min. Centrifuge the tube at setting 1 for 3 min.

Remove supernatant. Leave 0.5 cm on the beads. Do not let the beads dry out. Store beads on ice

7. Once the centrifugation run (step 5) is complete, retrieve your tube containing the protein
extract and immediately place on ice.
Move the sample to the cold room.
In the cold room: !!!!! Remove 2 x 300 l crude protein extract and place in 2 x 1.5 ml reaction
tube. Label the lid of the tube with the word Crude and your station number. Store in -20C
freezer located in the main lab.
In the cold room, transfer the supernatant into the 15 ml tube containing the Ni-beads. Label your
tube with tape and write your station number on the tape. Place the tube on the rotator in the cold
room.
Incubate the Ni-bead/cell lysate slurry for 1 h in the cold room.

8. During the wait time, prepare the column. Wash the column with 5 ml lysis buffer. Leave 0.5
cm buffer in the column.

9. Once the 1 h incubation time from step 8 is over, transfer the Ni-bead/protein extract slurry
into the column. Note, the column holds 5 ml.
Open the column and let the extract drain out of the column. During the time replenish with
addition Ni-beads/protein extract slurry, until all beads have been transferred into the column.
Collect the extract flowing through the column.
!!!!! Once all protein extract has passed through the column remove 2x 300 l from the flow-
through fraction and place in 2x 1.5 ml reaction tube. Label the lid of the tube with the word
Flow and your station number. Store in -20C freezer located in the main lab.
10. Use 10 ml additional lysis buffer to retrieve all Ni-beads from the 15 ml Flacon tube. Load
onto the column.

11. Wash Ni-beads with 10 mL wash buffer (do not forget to add the PMSF before using the
wash buffer.

M-32

12. Elute the protein into the 1.5 mL reaction tubes. Label the tubes fraction #1-#5. You will use
an elution buffer volume of 5 total and collect five 1 mL fractions.
For fraction one: Place the first 1.5 mL tube under the column. Add 1 mL elution buffer to the
column (please do not forget to add PMSF to the Elution buffer prior to use). Collect the entire 1
mL volume in the first tube. Close the lid of tube one and store on ice.
For fraction 2: Place tube 2 under the column, add 1 mL buffer to the column, elute 1 mL, and
save the fraction on ice.
Repeat this procedure for fractions 3-5.

13. Storage of the samples:
Samples for Bradford and SDS-PAGE
- Transfer 200 L from each fraction (tube #1-#5) into a separate 1.5 mL screw cap tube.
Label tubes Fraction#1-SDS Fraction#2-SDS etc.
You will save these fractions in the -20C freezer.

14. Storage of samples for gel permeation assay
- Add 0.4 ml glycerol to the remaining 0.8 ml of your fractions #1-#5. Mix well until glycerol is
completely mixed with fractions.

Label the lid of the 5 tubes with HisLDH #1, -#2, -#3, -#4, or -#5.

15. Store the five tubes in the 0C refrigerator.

Do not use tape to label the tubes!!!!!!

16. Close the bottom of your column, add 2 mL 1 M imidazole to the column, seal the top with
parafilm, and place the column in the collection container.

M-33
Experiment 2D: Determining Protein Concentration

On this day, you will determine the protein concentration, in mg/mL, of your crude extract, your
desalted ammonium sulfate fractions, and your pseudo-affinity purified LDH fractions.

Materials.
For Bradford Assays:
1. Concentrated Bradford reagent (Bio-Rad).
2. BSA at 0.2 mg/mL in 10 mM Tris-HCl, pH = 8.6.

To determine the yield of LDH, and so that you can determine the specific activity of the enzyme
at various steps in the purification procedure, you must determine the concentration of protein in
1) the crude bacterial extract your and 2) the first 4 fractions eluted from your Nickel affinity
column stored on ice.

Standard Curve table
Assay # Protein
Added
(g)
200
ug/mL
BSA
Stock
(l)
Sample
Type
10 mM
Tris-
HCl, pH
= 8.6
Bradford
Reagent
(l)
Final
Volume
(mL)
A
595
Measured
Protein
(g)
1 0 0 Blank 800 200 1.0
2 2 BSA 200 1.0
3 4 BSA 200 1.0
4 6 BSA 200 1.0
5 8 BSA 200 1.0
6 10 BSA 200 1.0


M-34

Sample preparation table
Assay # Sample
dilution
factor
Volume
added
(l)
Sample
Type
10
mM
Tris-
HCl,
pH =
8.6
Bradford
Reagent
(l)
Final
Volume
(mL)
A
595
Measured
Protein
(g)
1 100 10 Crude 200 1.0
2 100 50 Crude 200 1.0
3 100 10 Flow
Through
200 1.0
4 100 50 Flow
Through
200 1.0
5 1 10 HisLDH1 200 1.0
6 10 10 HisLDH1 200 1.0
7 1 10 HisLDH2 200 1.0
8 10 10 HisLDH2 200 1.0
9 1 10 HisLDH3 200 1.0
10 10 10 HisLDH3 200 1.0
11 1 10 HisLDH4 200 1.0
12 10 10 HisLDH4 200 1.0
13 1 10 HisLDH5 200 1.0
14 10 10 HisLDH5 200 1.0

1. You will set up tubes for the Bradford assay The BSA standards and your LDH fractions
should be diluted with 10 mM Tris-HCl, pH = 8.6. For your standard curve, use 0, 2.0, 4.0,
6.0, 8.0, and 10.0 g of BSA in a total volume of 0.8 mL.
REMEMBER THAT YOUR LDH FRACTIONS CONTAIN TRACES OF THE
POISON, 2-MERCAPTOETHANOL. WEAR GLOVES.
2. Set up tubes for your LDH fractions (use 10 mM Tris-HCl, pH = 8.6, as the diluent and be
sure that each tube contains a total volume of 0.8 mL). These fractions will have to be
diluted so that their A
595
values will fall in the range of the BSA standards. For the crude
bacterial lysate try using 10 L and 50 L of a 100-fold dilution (diluted into 10 mM Tris-
HCl, pH = 8.6). For your four Ni-Affinity fractions, try using 10 L of the undiluted fraction
and 10 L of a 10-fold dilution (diluted into 10 mM Tris-HCl, pH = 8.6).
PREPARE THE APPROPRIATE TABLE INCLUDING CALCULATIONS FOR
YOUR DILUTIONS IN YOUR NOTEBOOK BEFORE COMING TO THE
LABORATORY.
3. To start the Bradford assay, you will need to add 200 L of concentrated Bradford reagent to
each tube. Do so only after all samples have been prepared and assay can be performed on all
samples within the same amount of incubation time. Mix thoroughly and let each tube
incubate for at least 5 min (but no longer than 50 min) at room temperature. Record the A
595

value of each tube.

M-35
4. For the recombinant (His)LDH prepare a graph that shows the concentration of protein (in
mg/mL) in the crude extract, flow through fraction and fractions number.

5. Complete purification table. Using the obtained protein concentrations calculate Yield of
purification as a means to evaluate the success of the purification procedure

Fraction
Volume
(mL)

Concentration
of Protein
(mg/mL)
Purification (%)
(based on protein
concentration)
Crude Extract

Flow-through

Fraction 1
Fraction 2
Fraction 3
Fraction 4


M-36
Experiment 3: Analysis of LDH Purity: SDS-Polyacrylamide Gel Electrophoresis SDS-
PAGE

Day 8. SDS-PAGE Part A

On this day you will 1) pour two SDS polyacrylamide gels; 2) denature and electrophoresis
aliquots of your crude tissue homogenate, desalted ammonium sulfate fraction, and ''Peak'' LDH
fraction from both types of columns through these gels; and 3) fix and stain the gels. For details
of the procedures,
1. Pour a 12.5% acrylamide/0.1% SDS gel (either now or during step 2). Wait at least 20 minutes
before pouring a 5% acrylamide/0.1% SDS stacking gel and inserting the comb. Wait at least 20
min before removing the comb.
A. Assembling the Glass Plate Sandwich in the Casting Stand
1) Make sure the plates, spacers comb and casting stand gaskets are clean and dry before
proceeding. If they are greasy, wipe them with a Kimwipe wetted with 95% ethanol (DO
NOT USE A BROWN PAPER TOWEL - IT WILL SCRATCH THE GLASS PLATES),
from the wash bottles, until the plates are "squeaky" clean.
Assemble the glass-gel spacer sandwich on a clean surface. Lay the longer rectangular glass
plate down first, then place the two spacers along the short edges of the rectangular plate (see
figure below).

Top plate
Teflon Spacer

Teflon Spacer

Bottom glass plate

Next, place the shorter glass plate on top of the spacers so that the bottom ends of the spacers
and glass plates are aligned. At this point, the spacers should be sticking up above the long
glass plate about 5 mm.

M-37
2) Loosen the 4 "thumb & finger" screws on the clamp assembly. Stand it up so that the
screws are facing way from you. (Do not turn the two slotted screwdriver screws). The long
"notch" in the pressure plate should be at the bottom. The "ears" on the clamp assembly
should point up so that the clamp assembly sits flat on the lab bench top. Pick up the glass
plate sandwich. Firmly grasp it and gently slide it into the clamp assembly with the short
glass plate toward you. The thick acrylic (Plexiglas) "pressure plate" should be behind the
long glass plate (the glass plate farthest from you). Align the spacers and tap spacers and
glass plates down so they are flush with the lab bench. Gently tighten the top screws (only)
on the clamp assembly. Do not over-tighten! You will loosen them again in a minute.
3) Place the clamp assembly into the alignment position on the gel casting stand (see figure
below). There is one casting stand per two groups. But your glass plates will not be in the
stand for long, so do not worry about whether your plates are the first ones into the stand.

The clamp screws should face the square plastic "dome" on the casting stand (away from
you). Loosen the 4 thumb and finger screws to allow the plates and spacers to fall down and
sit flush against the base of the casting stand. The TAs will demonstrate the proper position.
Slide the Alignment Card (white card with green writing) between the glass plates to hold the
Casting slots
with removable
silicone gaskets
Alignment
slot

M-38
spacers in the proper position. There is one alignment card per two groups, but you will only
need it a moment. Position the spacers and tap the top of the spacers so they will slide down
and fit flush against the bottom of the casting stand. Hold the clamps at the base of the stand
and re-tighten the top two clamp screws. Use gentle but firm pressure! You are not putting
a tire on a car.
Failure to properly align the glass plates in this step can result in leaks while pouring the gel,
or buffer leaks during the run.
4) Leave the screws tightened. Remove the assembled glass plates, spacers and clamp
assembly from the gel-casting stand. Tighten the bottom 2 thumb and finger screws in the
clamp and check the tightness of the top screws. The screws should be tight enough to hold
everything firmly in place, but do not over-tighten.
CHECK FOR PROPER ALIGNMENT OF THE TWO PLATES AND SPACERS.
THE BOTTOM OF ALL THREE SHOULD BE FLUSH.

5) Put the clamp-glass plate assembly into one of the gel casting positions on the gel casting
stand
The clamps point in, toward the rectangular Plexiglas "tower" (see below). To attach the
clamp assembly in position, position the clamp assembly in the center of the casting stand.
Butt the thick acrylic pressure plate against the wall of the casting tower at the bottom, so the
bottom of the glass plates rest on the red rubber gasket.
6) Press down, and snap the acrylic plate underneath the overhanging "hook" of the casting
stand by pushing on the white part of the clamps. Do not push against the glass plates or
spacers, as this could break the plates. The sandwich is now ready for casting the gel. Two
"sandwiches" can be accommodated by each casting stand.
7) Fill the glass plates with water to test for leaks. If there is a leak, empty the water, dry the
plates, and reassemble the apparatus. If no leak is found, turn the apparatus on its side and
drain the water. Use a Kimwipe to wick out the last of the water. The inside does not need to
be absolutely dry. A little water will simply float on the top of the acrylamide solution you
will add.

M-39
B. Casting the Resolving Gel
The necessary stock solutions have been placed at one end of the laboratory bench.
CAUTION - acrylamide is a neurotoxin if absorbed through the skin and TEMED is
reputed to be carcinogenic. Wear gloves.

1) Place a "well-forming comb" into the top of the assembled glass plate sandwich (see
below). The top of the comb should be resting against the tops of the Teflon spacers. With a
ruler and marking pen, place a mark on the glass plate 1 cm below the teeth of the comb. This
is the level to which the top of the resolving gel will be poured. Remove the comb.

Before you pour the resolving gel (below), check to see that your setup is tight by filling it up
with deionized water.
2) DO NOT PIPETTE BY MOUTH. To prepare 10 mL of the solution used to cast 2
12.5% polyacrylamide resolving gels (A,B), combining all reagents given below in the flask
or test tube provided.
1.4 mL dH
2
O
2.5 mL l.5 M Tris HCl (pH 8.8); 0.4% SDS
5.6 mL acrylamide (37.5/1%)
500 l 0.75% APS (ammonium persulfate)
7 l TEMED

Swirl gently.
3) Read this step and step 4 below completely before proceeding.
Prior to initiating polymerization to the whole solution, remove 1.5 mL of your 12.5%
resolving gel and place into an Eppendorf tube and add 7 l TEMED to this than upend it
once to mix and quickly transfer .75 mL in to each of the gel casting slots without introducing
air bubbles followed by a gently added layer of H
2
O. This will seal up the bottom of the gel
and solidify in about a minute so that no leakage will occur. Now you can remove the excess
H
2
O from the cast and move on to adding 7 l TEMED to you remaining resolving gel and
gently swirl.
TEMED catalyzes the crosslinking of the polyacrylamide chains to one another, resulting in
polymerization. After the TEMED is added, you must proceed quickly. You have
approximately 5 minutes to pour the resolving gel before the solution will polymerize.
4) Using a Pasteur pipette, add the solution along one side of the slab gel apparatus and fill up
to the pen mark you made on the glass. Avoid making air bubbles as they may be trapped
when the polyacrylamide solidifies. If any bubbles form, tap the glass plates gently until they
float to the surface.
5) Overlay the gel by gently adding a small amount of dH
2
O with a Pasteur pipet to the top
of the gel. BE CAREFUL! IT MUST LAYER ACROSS THE TOP OF THE GEL
WITHOUT MIXING WITH THE ACRYLAMIDE SOLUTION. Because the water is
less dense than the acrylamide solution (which has lots of chemicals dissolved in it) It will
float on the acrylamide solution and spread out to cover the top of the gel solution. As the gel

M-40
polymerizes, the dH
2
O will flatten the top of the gel and prevent localized drying and
shrinking of the gel surface.
6) Place the slab gel in a location where it will not be disturbed. Polymerization should be
complete in 30 minutes.
Remove the water overlay from the top of the gel with blotting paper or a Kimwipe.
7) Pour a 5% acrylamide; 0. 1% SDS stacking gel above the resolving gels.
Mix:
3.9 mL dH
2
O
2.4 mL 0.5 M TrisHCl (pH 6.8); 0.4% SDS
2.3 mL acrylamide (37.5/1%)

Just before pouring, add:
1.5 mL 0.75% APS (ammonium persulfate)
5 l TEMED

8) Pour the stacking gel solution over the resolving gel.
9) Insert comb.
10) Let stand at least 20 min undisturbed before removing the comb.
11) Prepare the samples for electrophoresis as table indicates: heat 5 min at 90 C, and
microfuge for 1 min. Load the 10 l volume for each sample, and run at 25 mA/gel for
approximately 30-40 min with a TA or coordinators assistance.
12. After gel has completed running carefully stop the power supply and turn it off. Gently
pull out Inner cooling core from running chamber dumping out excess buffer.
13. The TA or coordinator will demonstrate how you should remove gels from the stand
appropriately.
14. Stacking gel gets are removed and resolving gel goes into the Coomassie staining
solution on a rotating table until the next session.

M-41

Gels A and B: Fill out this table before coming in to class
Gel Tube
No./gel
Sample Sample
Conc.
(Bradford)
(mg/mL)
Amount
of
sample
to use
for
stock
(g)
Volume
of
sample
(l)
Volume
of H
2
O
(l)
Volume
of 6x
Sample
Buffer
(l)
Total
sample
volume
(l)
Total
volume
to load
on to
the gel
(l)
A 1 None
A 2 Standard 10
A 3 Crude
Super.
240 10 60 10
A 4 Desalted
AmSO4
240 10 60 10
A 5 CB Peak 60 10 60 10
A 6 Standard 10
A 7 Crude
Super.
120 10 60 10
A 8 Desalted
AmSO4
120 10 60 10
A 9 CB Peak 30 10 60 10
A 10 None
B 11 Standard 10
B 12 Induced Experiment 2b 20
B 13 Uninduced Experiment 2b 20
B 14 Standard 10
B 15 His-LDH1 30 10 60 10
B 16 His-LDH1 60 10 60 10
B 17 His-LDH2 30 10 60 10
B 18 His-LDH2 60 10 60 10
B 19 His-LDH3 30 10 60 10
B 20 His-LDH3 60 10 60 10


M-42
Experiment 3B: SDS-PAGE

Day 9. Destain SDS gels continued
One member will retrieve you groups gel from shaker in order to continue processing your gels
from day 8. This process can be completed in between doing experiment 5.
1. Pour Coomasie stain back into its 50 mL Falcon tube and add about 50 mL of H
2
O to
your gel. Swirl around H
2
O and dump into sink with water running.
2. Add enough destaining solution to the gel to just cover the top and return box to the
shacker for 30-60 minutes.
3. After this amount of time has passed pour used destain into proper waste container and
refill destaining box with fresh destaining solution. Twist up 2 Kimwipes and place beside
your gels into the solution being careful not to place them on top of the SDS gels.
4. Place box back on to shaker until next session.


M-43
Experiment 4: Yield and Specific Activity of LDH

Days 9/10.

On this day you will measure the activity of these fractions by monitoring the formation of
NADH spectrophotometrically at 340 nm. By combining these results with the protein
concentrations of your crude extract, desalted ammonium sulfate fraction, and pseudo-affinity
purified LDH fractions (determined on Day 7), you will be able to determine your yield of
purified LDH and the magnitude of its specific activity increase during purification.

Materials

Materials on ice:

1. Thaw one each: Crude, Desalt and CB fraction #1 on ice.

2. Recombinant Lactate Dehydrogenase purified from bacteria, it should be kept
on ice.

This concentrated LDH may be diluted into cold 10 mM Tris-HCl, 1 mg/mL BSA, pH =
8.6 immediately before you need it. Listen for directions from the lab coordinator.
Keep this solution on ice in case you need it.

3. 10 mM Tris-HCl, 1 mg/mL BSA, pH = 8.6. (For diluting your LDH fractions)

Materials Initially at room temperature:

1. LACTATE STOCK: 120 mM lithium lactate in 10 mM Tris-HCl, pH = 8.8.

2. NAD
+
STOCK: 12 mM NAD
+
in 10 mM Tris-HCl, pH = 8.8.

3. BICARBONATE STOCK: 0.5 M NaCl containing 18 mM NaHCO
3
.

4. Plastic cuvette.

5. Plastic cuvette mixer ("plumper").

Procedure

Activity Assays - Recombinant LDH

1. Before beginning, thaw your Crude, Desalt and CB fraction #1 on ice.

2. Take your pipettes and tips to the spectrophotometer.


M-44
The NAD
+
-dependent conversion of L-lactate to pyruvate offers a convenient assay for the
presence of LDH {L-lactate + NAD
+
Pyruvate + NADH + H
+
}. The formation of NADH can
be monitored by its absorbance at 340 nm. Stock solutions of NAD
+
are much more stable than
those of NADH because NADH is readily oxidized by air.

3. To become familiar with the procedure, you should perform several assays with the
Recombinant LDH
a. Make sure the visible lamp on.
b. For the DU-640, Click on ''KINETICS/TIME'' at the upper left of the main window. For
the DU-800, go to the METHODS menu, chose CREATE/EDIT METHOD.
c. Set the analytical wavelength to 340 nm.
d. Set the interval time to 1.5 sec (so that a reading will be recorded every 1.5 sec).
e. Set the total time of the assay to 40 sec.
f. Set the Factor setting to 1.0.
g. Set the Read Average Time to 0.1 second.

2. Assay Procedure.

Make the assay mixture just before running the assaying in the cuvette! Do this by
following the directions below.

a. To a NEW plastic cuvette in position in the spectrophotometer, add 0.6 mL of the
LACTATE STOCK, 0.4 mL of the NAD
+
STOCK, and 0.2 mL of the
BICARBONATE STOCK (What are the final concentrations of lactate, NAD
+
, NaCl,
and bicarbonate in the 1.2 mL assay volume?) Mix these components with the plumper
in the spectrophotometer.
b. Shut the lid. Click on ''Blank'' in the lower left of the screen to record the background
absorbance of buffer plus substrate in the absence of enzyme.
c. Click on ''Read Samples.'' This calls up the ''Insert sample(s)" dialogue box.
d. Addition of the enzyme:
1) Open the lid of the spectrophotometer.
2) Place the plumper in the cuvette (leave the cuvette in the spectrophotometer).
3) Slowly draw 10 L of enzyme (1.0 mg/mL) into a yellow tip.
4) Carefully remove outside droplets with a KimWipe.
5) Inject the diluted enzyme into the cuvette.
6) Mix the contents of the cuvette by moving the plumper up and down three or four
times.
7) Remove the plumper.
8) Close the spectrophotometer lid.
9) Click Start.
Steps 5) to 9) should be performed rapidly. Proper use of the plumper will quickly mix
the enzyme with the assay mix so that you can begin collecting data as close to the true
time = zero point as possible. It might help if one partner operated the pipetman and the
other operated the computer mouse.

Blot off your plumper with a Kimwipe before going on to your next assay.

M-45

e. On the DU-640, at the conclusion of the scan, the ''Tabulating Data" message box will
appear briefly, then disappear.
f. On the DU-640, Click on "Autoscale" in the center of the screen to better display the data
trace. On the DU-800 Autoscale will run automatically.
g. On the DU-640, click "annotate," then label the spectrum and print it using the print
command in the upper right comer of the screen. On the DU-800 double click on the
graphing space to make an annotation box appear.
h. To obtain an estimate of the rate that the A
340
increases in units of A
340
/min, for the DU-
640, click on ''Rate.'' Click on "Autoscale." For the DU-800, click Mode and click
Rate on the drop down menu. Leave the "Initial Time" set to 0.0000. Set the Final
Time to 5 sec, then 10 sec, then 20 sec, then 30 sec. For each "Final Time" value,
record in your notebook (and on your printed plot) the A
340
/min and variance values.
For how long is the rate linear? What is your best estimate of the true initial rate in
A
340
/min? What is the variance of this rate? The rate of the A
340
increase gradually
slows as pyruvate accumulates (feed back inhibiting the enzyme) and as the enzyme
denatures.

The rate of change in the A
340
should be about 0.300 / 20 seconds.

If the rate is significantly more or less than this rate, change the dilution of the of enzyme

i. Repeat the activity measurement with a fresh sample and vacuumed out cuvette. (Wash
and thoroughly dry the plumper between samples). To reset the instrument, click "exit"
to leave the ''rates'' menu, then click "SaveClear" in the upper right. On the DU-640,
uncheck the "Save Results" box, then click OK. For the DU-800, click on Clear
Results and Dont Save Data. Go back to step b, above, and proceed from there
(obtaining a fresh background absorbance value with fresh assay mix in the cuvette).
j. Determine the initial activity, v
o
, in the cuvette, for each sample. From these values,
calculate the activity of your UNDILUTED enzyme solution. [To do this, convert the
initial rate (A
340
/min) to moles of product formed per min per mL (the activity should
be expressed as "units/mL," where 1 unit = 1 mole of product formed per minute)].
Remember that the extinction coefficient for NADH at 340 nm is 6.22 x 10
3
M
-1
cm
-1
and
that you are diluting 10 L of diluted enzyme into a total assay volume of 1.2 mL.
k. Your Recombinant LDH was diluted around 10-20 fold from a concentrated stock
solution that you measured previously. Use this information to calculate the specific
activity of the Recombinant LDH in your concentrated stock solution.
l. Recall that V
max
= k
cat
E
o
and that the molecular weight of LDH is 140,000. Assuming
that your value for V
o
is approximately 85% of V
max
, estimate the turnover number (k
cat
)
of hog muscle LDH for lactate (in sec
-1
) under the conditions of your assays. Estimate
the precision of your estimate (i.e., report k
cat
= (x x) sec
-1
). Look in Appendix for
Experiments 5/6/7 for help in calculating precision.

3. Activity Assays - Your LDH fractions from chicken breast muscle.
a. Place one of your frozen 1 mL aliquots of Fraction 1 from the Cibacron Blue Column on
ice to thaw.

M-46
b. Once you have become proficient with the enzyme assay, measure the initial activity for
your 1) crude tissue homogenate; 2) desalted ammonium sulfate fraction; and 3) the first
fraction that was eluted from the Cibacron Blue column by NADH (unless you found a
higher protein concentration in the second fraction, if so, then measure the initial activity
of the second fraction); and 4) Recombinant LDH peak sample.
YOU WILL HAVE TO DILUTE THESE FRACTIONS. Otherwise, the rate of the
A340 increase will slow too rapidly to permit the initial activity to be estimated with any
accuracy. Ideally, you should have a slope of ~ 0.3 A
340
units per minute over the time
interval 0 - 20 sec. Try making a 50-fold dilution of each fraction. The LDH in these
dilutions may denature rapidly. Therefore, make each dilution IMMEDIATELY
before use. To do this, dilute 5 L, of your LDH fraction into 240 L of ice-cold 10 mM
Tris-HCl, pH = 8.6 (If a different dilution is required still use 5 l protein and adjust the
amount of buffer). AFTER the lactate, NAD
+
, and bicarbonate stocks have been mixed
in the cuvette and the spectrophotometer has been calibrated. Quickly run sample until
you can accomplish a similar rate at least 3 times for each sample type, the average will
be used to fill out your activity table.

If the slope (rate) is less than 0.09, try using your sample without dilution.


M-47
4. Protein Purification Table and Cibacron Blue Elution Profile.

a. On the elution profile that you drew in part 7 of Day 3, plot the activity (in units/mL) that
is present in each fraction that was eluted from the Cibacron Blue column by NADH (you
have determined the activity for only the first four fractions).
b. Calculate the specific activity of LDH in each of your fractions: the crude tissue
homogenate, the desalted ammonium sulfate fraction, and the first four fractions that
were eluted from the Cibacron Blue column by NADH. Construct a protein purification
table that shows how the following parameters changed during each step in the
purification procedure: 1) Total Amount of Protein (mg); 2) Total Amount of Activity
(in units); 3) Yield of Activity (in percent, with 100% being the amount of activity that
was present in the crude homogenate); 4) Specific Activity (in units/mg); 5) 'Purification
Factor (the ratio of the specific activity to the specific activity of the crude homogenate).

5. Store your remaining LDH fractions at 4C (including the tube of fraction 1 that you
thawed today). You will use the fraction with the highest specific activity (usually this will
be fraction 1) to characterize the kinetic properties of chicken breast muscle LDH during
days 5 and 6, and you will determine the purity of all of your fractions by SDS- PAGE
during days 7 and 8.

SAMPLE PROTEIN PURIFICATION TABLE

Fraction
Volume
(mL)

Concentration
of Protein
(mg/mL)
Total
Amount
of Protein
Activity
(units/mL)
Specific
Activity
(units/mg)
Total
Amount of
Activity
(Units)
Yield of
LDH
Purification
Factor
(-fold)
Crude Extract 43 20.0 860 220 11.2 9,540 (100%) (1 )

Desalted
Ammonium
Sulfate Fraction
3.8 39.0 148 480 12.4 1,830 19.2% 1.11

Peak NADH
fraction from
Cibacron Blue
column
5.0 1.25 6.25 240 190 1,190 12.5% 17.0

Peak
Recombinant
LDH fraction
0.8 3.25 3.25 320 98.46 320 -


M-48
Experiment 5: K
M
and k
cat
of uninhibited LDH

Days 11/12

On this day you will determine the K
M
for lactate of your purified LDH. You will accomplish
this by measuring the activity of your LDH in the presence of different concentrations of lactate.

Materials (all initially at room temperature).

2. NAD
+
STOCK: 12 mM NAD
+
3
.
4. 10 mM Tris-HCl, pH = 8.8 (for diluting the lactate stock).
5. 10 mM Tris-HCl, BSA pH = 8.6 (for diluting your LDH fraction)
6. Plastic cuvette.
7. Plastic cuvette mixer (plumper).

Procedure.

1. Before beginning,
a. Place one of your frozen 1 mL aliquots of Fraction 1 from the Cibacron Blue Column on
ice to thaw. If your Recombinant LDH had higher activity than your purified sample use
it for the following procedures.
b. Place a 10 mL aliquot of your 10 mM Tris-HCl, BSA pH = 8.6 buffer on ice.

2. You will perform the same assay that you used on Day 5, except you should only measure
the rate at 20 sec, rather than at the multiple times you used on Day 5. Use your ''Peak
Fraction" of LDH. Use the same dilution of the ''Peak Fraction" that worked well on Day 5.
Remember that you should have a slope of ~0.3 A
340
units per minute over the time interval 0
- 0.3 min. REMEMBER TO MAKE A FRESH DILUTION OF YOUR ENZYME FOR
EACH SET OF ASSAYS AND TO MAKE THESE DILUTIONS IMMEDIATELY
BEFORE USE.

3. Perform the assay in the presence of the following concentrations of lactate.

5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM and 60 mM.

a What was the concentration of lactate in the assay buffer on Day 4? Decrease the lactate
concentration present in this buffer by decreasing the amount of LACTATE STOCK
added per assay, replacing the missing volume with 10 mM Tris-HCl, pH = 8.8.
PREPARE THE APPROPRIATE TABLE IN YOUR NOTEBOOK BEFORE
COMING TO THE LABORATORY.

b. Perform one assay at each concentration of lactate.
Perform a second assay at each concentration of lactate.

M-49
If any of the duplicates differ significantly, perform a third assay at that concentration of
lactate.

4. For each assay, calculate the initial rate, v
o
, of product formation in the cuvette at each lactate
concentration (remember that you are using 1.2 mL of assay buffer in a cuvette that has a
path length of 1 cm).

5. Use a DIRECT LINEAR PLOT to estimate the K
M
and V
max
of your LDH for lactate under
these assay conditions. These plots must be drawn carefully. USE A SHARP-TIPPED
WRITING IMPLEMENT, A GOOD STRAIGHT EDGE, AND A SHEET OF HIGH
QUALITY GRAPH PAPER. Carefully mark the intersection points of all of the lines as you
draw the lines! From these intersection points, estimate the uncertainty in your K
M
and V
max

values. HAVE YOUR TA AND/OR INSTRUCTOR CHECK YOUR PLOTS BEFORE
YOU LEAVE THE LABORATORY!

For an explanation of direct linear plots and examples see Appendix C.

6. Plot your data in the form of a Lineweaver-Burk plot. Draw lines that correspond to the K
M

and V
max
values that you obtained from the Direct Linear Plot. What can you conclude about
the relative merits of the Lineweaver-Burk and Direct Linear plots for analyzing data? When
is it appropriate to employ a Lineweaver-Burk plot?

7. Given that the molecular weight LDH is 140,000 MW, and that V
max
= k
cat
E
o
, calculate the
turnover number of your LDH for lactate (in sec
-1
) under the conditions of your assays.
What is the precision of your estimate (i.e., k
cat
= (x x) sec
-1
)?

8. Store the remaining portion of your Peak sample at 4C.


M-50
Experiment 6: K
M
and k
cat
of inhibited LDH

Day 13.

On this day you will characterize the inhibition of chicken breast muscle LDH by sodium oxalate
and determine the dissociation constant of oxalate for LDH. You will accomplish this by
measuring the activity of your LDH in the presence of a constant amount of oxalate and different
concentrations of lactate.

Materials (all initially at room temperature).

2. NAD
+
STOCK: 12 mM NAD
+
3
.
4. OXALATE STOCK: 3.0 mM oxalate acid (disodium salt) in 10 mM Tris-HCl, pH = 8.8.
5. 10 mM Tris-HCl, pH = 8.8 (for diluting the lactate stock).
6. 10 mM Tris-HCl, BSA pH = 8.6 (for diluting your LDH fraction)
7. Plastic cuvette.
8. Plastic cuvette mixer ("plumper").

Procedure.

1. Before beginning,
a. Place another of your frozen 1 mL aliquots of Fraction 1 from the Cibacron Blue Column
on ice to thaw. If your Recombinant LDH had higher activity than your purified sample
use it for the following procedures.
b. Place a 10 mL aliquot of your 10 mM Tris-HCl, BSA pH = 8.6 buffer on ice.

2. You will perform the same assay that you used on Days 5 and 6, EXCEPT THAT EACH
ASSAY SHOULD CONTAIN 0.25 mM OXALATE. Use your "Peak Fraction" of LDH
(if your purification didn't work, you will be provided with a 50-fold dilution of 10 mg/mL.
hog muscle LDH). Use the same dilution of the "Peak Fraction" that worked well on
Days 5 and 6. Adjust the dilution fold until you obtain a rate with 60 mM lactate that is
~0.3, as on the previous days. Make at least 100 L of this dilution, using a minimum of
10 L of your enzyme. Use this dilution of enzyme to for all of the first set of assays.

3. Perform the assay in the presence of the following concentrations of lactate.

10 mM, 20 mM, 30 mM, 40 mM, and 50 mM.

a. As on Day 5, vary the lactate concentration by varying the amount of LACTATE
STOCK used per assay. Because each assay will be performed in the presence of 0.25
mM oxalate, you will replace the "missing" volume with a combination of 10 mM Tris-
HCl, pH = 8.8 and OXALATE STOCK. PREPARE THE APPROPRIATE TABLE IN
YOUR NOTEBOOK BEFORE COMING TO THE LABORATORY.


M-51
b. Perform one assay at each concentration of lactate.
Perform a second assay at each concentration of lactate.
If any of the duplicates differ significantly, perform a third assay at that concentration of
lactate.

4. For each assay, calculate the initial rate, v
o
, of product formation in the cuvette at each
lactate concentration (remember that you are using 1.2 mL of assay buffer in a cuvette that
has a path length of 1 cm).

5. Use a DIRECT LINEAR PLOT to estimate the K
M
(apparent) and V
max
(apparent) of your
inhibited LDH for lactate under these assay conditions. These plots must be drawn carefully.
USE A SHARP-TIPPED WRITING IMPLEMENT, A GOOD STRAIGHT EDGE,
AND A SHEET OF HIGH QUALITY GRAPH PAPER. Carefully mark the intersection
points of all of the lines as you draw the lines! From these intersection points, estimate the
uncertainty in your K
M
(apparent) and V
max
(apparent) values. HAVE YOUR TA AND/OR
INSTRUCTOR CHECK YOUR PLOTS BEFORE YOU LEAVE THE
LABORATORY!

6. Compare the K
M
(apparent) and V
max
(apparent) determined in the presence of 0.25 mM
oxalate with the K
M
and V
max
values determined on Day 6 in the absence of inhibitor. Given
the precision of your data, is V
max
(apparent) significantly different from V
max
? Is
K
M
(apparent) significantly different from K
M
? Explain your answers.

7. Plot your data in the form of a Lineweaver-Burk plot ON THE SAME LINEWEAVER
BURK GRAPH THAT YOU DREW FOR YOUR UNINHIBITED DATA ON DAY 5.
Draw lines that correspond to the K
M
(apparent) (K
M
app
) and V
max
(apparent) (V
max
app
) values
that you obtained from your Direct Linear Plot, above. What can you conclude about the
relative merits of the Lineweaver-Burk and Direct Linear plots for analyzing data? When is
it appropriate to employ a Lineweaver-Burk plot?

8. Is oxalate a competitive, non-competitive, uncompetitive, or mixed inhibitor of the lactate +
NAD
+
pyruvate + NADH reaction catalyzed by chicken breast muscle LDH? Explain
your reasoning.

9. Assuming that oxalate is a competitive inhibitor, calculate the dissociation constant (K
I
) of
oxalate for chicken breast muscle LDH. Report the precision of your estimate.

10. Store the remaining portion of your Fraction 1 from the Cibacron Blue Column at 4C.


M-52
Experiment 1-7 Format for Lab Report
(330 points)

I. Title Page

II. Objectives
A. Write a paragraph that briefly gives an overview of the experiment and explains its
purpose.

III. Procedures
A. Purification of LDH from chicken
1. Homogenization in the presence of PMSF and 2-ME.
2. Precipitation with ammonium sulfate.
3. Resuspension and desalting.
4. Chromatography with Cibacron Blue.
B. Purification with Nickel affinitychromatography
1. Expression (Graph)
2. Homogenization
3. Metal ion chromatography

B. Kinetic Characterization
1. Determination of K
M
, V
max
, and k
cat
with lactate as the substrate (Explain the assay
and why you measured the absorbance at 340 nm).
2. Determination of the precision of the values of K
M
, V
max
, and k
cat
.
3. Determination of mode of inhibition of oxalate, including determination of the
apparent K
M
(K
M
app
), V
max
app
, and K
i
with oxalate.
4. Determination of the precision of the values of K
M
app
, V
max
app
, and K
i
with oxalate.
5. Comparison LDH from chicken and bacteria

IV. Results
A. Purification
1. Observations during the various steps.
2. Elution Profiles.
a. Plot of A
280
, activity, and protein concentration versus fraction number for
chicken and recombinant LDH.
b. Bradford data for estimating the protein concentrations.
c. Raw activity data plus calculations of activity and specific activity. Include
raw activity plots for your chicken LDH (crude homogenate, desalted, purified
enzyme) fractions, and include the raw data obtained with the recombinant
LDH. Estimate the Specific Activity and k
cat
of the chicken and recombinant
LDH under your assay conditions.
3. Purification tables - with explanations
4. SDS-PAGE - Sketch of gels.

B. Kinetic Characterization
1. Raw data plots

M-53
2. Table of s
o
and v
o
values with and without inhibitor
3. Direct Linear Plots
4. Estimates of K
M
, K
M
app
, V
max
, V
max
app
, k
cat
, and K
i
, with precisions.
5. Lineweaver-Burk plots of data points, with lines drawn using the K
M
, V
max
, K
M
app
,
and V
max
app
values obtained from the direct linear plots.

V. Discussion (four pages, maximum).

A. Purification
Discuss the data in the purification table (e.g., specific activity, yield, purification factor)
in the context of the SDS-PAGE data and the overall purification procedure.
Compare (specific activity, yield, purification factor) of the two different purification
assays

B. Kinetic Data
Discuss the K
M
, K
M
app
, V
max
, V
max
app
, k
cat
, and k
i
values and the precision of these values.
Are K
M
app
and V
max
app
significantly different from K
M
and V
max
? Explain your answer.
How do the Specific Activity and k
cat
values of your chicken LDH compare with the
values that you calculated for the recombinant LDH? What type of inhibition is observed
with oxalate? Explain your answer. Discuss the advantages and disadvantages of the
Direct Linear and Lineweaver-Burk plots.

M-54

BCH 102 LABORATORY REPORT
Experiment #8: Crystallization of Proteins

Student Name (Last, First)

Date Received:

I. Objective/Introduction
a. Brief description of crystallography and objectives.

II. Procedures
a. Step by step procedure.

III. Results/Data
a. Indicate your fraction 1 activity, concentration, and whether you used NADH or lactate in
the crystallization conditions.
b. Include a copy of your observations.
c. Include any sketches you made of crystals.

IV. Discussion
a. What is the best condition (pH, salt, precipitant) for the crystallization of your protein
sample?
b. What is function of salt and precipitant for protein crystallization. Which conditions
appear to be best based on total lab data?
c. What conclusions can you make based on the fraction 1 specific activities, concentration,
and the addition of either lactate or NADH?
d. Why would you add NADH into your protein sample for crystallization?
e. What are the reasons you may not crystallize the sample?


M-55
Rubric for BCH-102 LABORATORY REPORT
Experiment #9: Protein crystallization

Student Name (last, first):

Date received:

Points received/Points Possible

Introduction: ____/5 pts

Objective: ____/2pts

Procedures: ____/5pts

Results/Data: ____/12pts

Discussion: ____/36pts

a. ____/12 pts What is the best condition (pH, salt, precipitant) for the crystallization of your
protein sample?

____/8 pts What is function of salt and precipitant for protein crystallization. Which
conditions appear to be best based on total lab data?

____/8 pts Why would you add NADH into your protein sample for crystallization?

____/8 pts What are the reasons you may not crystallize the sample?

____ /60 pts total points


M-56


M-57
Experiment 8: Molecular Biology - The transformation, purification and restriction
enzyme analysis of plasmid DNA.

OUTLINE OF THE EXPERIMENT

Day 1 Prepare two Ampicillin-containing, LB agar plates.

Day 2 Generate competent E. coli and Transformation of plasmid DNA
Preparation of chemically competent E. coli
Grow E. coli culture
Make the cells competent
Transform plasmid DNA into the competent E. coli.
Incubate the plates overnight.
Plasmid preparation
Grow transformed cells in ampicillin supplemented medium for Day 3

Day 3 Transformation efficiency and plasmid preparation
Determine transformation efficiency (count colonies)
Purify plasmid DNA using a QIAprep Spin Miniprep Kit
Determine the concentration and purity of the DNA

Day 4 Restriction analysis of DNA
Conduct restriction enzyme reactions
Analyze size of fragments using agarose gel electrophoresis

INTRODUCTION

Plasmids

Definition: A circular, double-stranded DNA that replicate within a cell independently of the
chromosomal DNA. They are extrachromosomal. They are ubiquitous in prokaryotes and have
also been identified in a number of eukaryotes. Plasmids are used in recombinant DNA research
to transfer genes between cells.
In general, bacterial plasmids can be classified into two groups on the basis of the number of
genes and functions they carry. One group, the larger plasmids, are DNA molecules of around
100 kilobase (kb) pairs, which is sufficient to code for approximately 100 genes. There are
usually a small number of copies of these plasmids per cell, so their replication must be precisely
coordinated with the replication of the host chromosome, and the cell division cycle. The
plasmids in the second group are smaller in size, about 6-10 kb pairs. These plasmids may
harbor 6-10 genes, and are usually present in multiple copies (10-20 per host cell).
Plasmids have been identified in a large number of bacterial genera. Some bacterial species
harbor plasmids with no known functions, which are termed cryptic plasmids. These plasmids
have no apparent effect on the phenotype of their host cell, and have no genes other than the ones
needed to replicate itself, and to spread to other cells. Other plasmids carry genes which can
confer, for example, antibiotic resistance to their host cell. The host range of a plasmid is usually

M-58
limited to closely related bacterial genera. However, other plasmids are much more
promiscuous, and have a much broader host range.
The functions specified by the genes on bacterial plasmids are usually specialized in nature.
They do not carry genes required for the survival or growth of the host cell under normal
circumstances, since the host bacteria are viable without a plasmid when the cells are cultured
under conditions that do not select for plasmid-specified gene products, like antibiotic resistance
genes. Plasmids introduce specialized functions to host cells, which provide versatility and
adaptability for growth and survival. Plasmids, which confer antibiotic resistance (such as
ampicillin resistance; R plasmids), have been extensively characterized because of their medical
importance.
Plasmids played a seminal role in the spectacular advances in the area of genetic engineering.
Individual genes can be inserted into specific sites on plasmids, the plasmids placed back into
bacterial cells by the process of bacterial transformation, and the cells with their engineered
plasmids grown to large numbers in culture. In this way, genes can be produced for further
study, and the genes for rare protein products can be expressed in bacterial cells to high levels,
making them available for study or use medically. Engineered plasmids that have been
genetically altered, such as by the insertion of an antibiotic resistance gene, are said to contain
recombinant DNA because the original genetic composition of the plasmid has been
artificially altered.
Transformation modifies the genotype of the recipient cell (usually a prokaryotic cell) by
introducing DNA from another source. During transformation, the DNA is taken up from the
surrounding medium, and usually becomes a circular, extrachromosomal passenger DNA, which
expresses new genetic information. In specialized cases, the plasmid DNA can be engineered to
integrate it into the host cell DNA, where it is replicated as a part of the host cell chromosome.
Transformation occurs naturally, and the resulting uptake of foreign DNA by the cell is not
typically considered recombinant DNA. However, transformation can be induced by treating
bacterial or eukaryotic cells in a number of specific ways. These treatments are said to induce
transformation "competency" in the cells, which is the ability to take up, replicate, and express
foreign DNA.
Bacteria can be made competent by "shocking" them, which involves cooling the bacteria in
a calcium chloride solution at 0C, then quickly heating the solution to approximately 47C for
90 seconds. Too long of a heat treatment will denature the cell membrane, killing the bacteria.
The calcium chloride ions neutralize the repulsion between the negatively charged phospholipid
head groups in the cell membrane and the negatively charged phosphate groups on the DNA.
The quick heat shock creates a thermal gradient, which creates an inflow of solution into the cell,
allowing the DNA (such as plasmids) in the solution to enter the cell. The plasmid DNA is
replicated, the genes on the plasmid are expressed and the bacterial cell becomes genetically
modified.
Cells become the most competent when they are harvested from the growth medium and
during their mid-log phase before undergoing the transformation procedure described above. In
a growing couture of E. coli cells, competent cells are most easily made when they are grown to
an absorbance of 0.25, measured using a standard spectrophotometer set to 660 nm (visible
light).


M-59
Restriction Analysis

A DNA restriction endonuclease (also called a restriction enzyme) is an enzyme that
recognizes a particular sequence of DNA bases in a double stranded DNA, and catalyzes the
cleavage of both strands of the DNA. In restriction analysis of recombinant DNA, the DNA is
treated with one or more restriction enzymes to verify that a piece of DNA has been successfully
inserted into the plasmid. A restriction map, is a map of the restriction endonuclease cleavage
sites in a plasmid DNA. Therefore, restriction endonuclease of a potential recombinant plasmid
can indicate the relative location and size of an inserted DNA.

Restriction enzymes (or restriction endonucleases) cleave DNA in a very specific fashion.
Type II restriction enzymes, most commonly used for DNA analysis and genetic engineering,
each have a unique, small, nucleotide sequence at which they cleave a DNA molecule. A
particular restriction enzyme will cleave DNA at that recognition sequence and nowhere else.
The recognition sequence is often a six base pair palindromic sequence (the top DNA strand
from 5' to 3' is the same as the bottom DNA strand from 5' to 3'), but others recognize four or
even eight base pair sequences. For example, the recognition site for HindIII is:

5 - NNNNAAGCTTNNNN- 3
3 - NNNNTTCGAANNNN- 5

where N represents any nucleotide of a double stranded DNA, and the bar shows where the
palindromic sequence begins to repeat in the other DNA strand.

Restriction enzymes can also differ in the way they cut the DNA molecule. Some enzymes
cut in the middle of the recognition sequence, resulting in a flush, or blunt end cut which
seperates the two halves of the palindrome. For example, Sma-1 cuts the restriction
endonuclease recognition site CCCGGG. between the Cs and Gs (e.g. CCC^GGG) to produce a
blunt end cut in the DNA. The sequence of only one strand (arbitrarily defined as the top
strand) is shown here. You can easily guess the sequence of the complementary, bottom strand.

Other enzymes cleave in a staggered fashion, resulting in DNA products that have short
single-stranded regions at their ends. These single stranded regions are sometimes called
overhangs. The overhangs are usually two or four nucleotides in length at each end. These
single stranded ends are also called cohesive ends, since the single stranded ends can reanneal
through complementary base-pairing if the temperature is low enough and the monovalent salt
concentration high enough to make the short hybrids stable. For example, the HindIII cleavage
site above is cleaved by the enzyme to produce staggered, cohesive ends as shown below:

5' - AAGCTT- 3' 5 - A AGCTT- 3
3' - TTCGAA- 5' 3 - TTCGA A- 5

At 4 C and 150 mM NaCl, ligase can join these ends in an overnight incubation. The hybrid
need be stable only long enough for some of the molecules to be ligated together.


M-60
A common use for restriction enzymes is to generate a characteristic series of DNA
fragments which can serve as a "fingerprint" of a particular double stranded DNA molecule like
a plasmid DNA. Because of the sequence specificity of restriction enzymes, these enzymes can
cut DNA into discrete fragments, which can be resolved by gel electrophoresis. This pattern of
DNA fragments generates a "DNA fingerprint,". Each DNA molecule typically has a unique
fingerprint. Other restriction enzymes can be used to further map the structure of a particular
DNA molecule. The location of these restriction enzyme cleavage sites on the DNA molecule
are compiled to create a restriction enzyme map. These maps are very useful for identifying
and characterizing the DNA of viruses, plasmids, cDNA, and genomic DNAs.

In this laboratory, you will use restriction enzymes to generate DNA profiles for plasmid DNA.

Genetic Engineering

Recombinant DNA (rDNA) is DNA that has been genetically altered in vitro. DNA can be
cut into shorter fragments with restriction enzymes, and these fragments ligated into new DNA
molecules, which do not exist in nature. By looking at the size of the fragments created by
restriction enzymes, investigators can determine the new arrangement of DNA fragments. These
techniques have been used to analyze genetic structures in fetal cells and to diagnose certain
blood disorders, such as sickle cell anemia.

M-61
Experiment 8a: Culturing of E. coli

Day 15

METHODS AND PROCEDURES

A. Make two Ampicillin containing, LB agar plates for platting your transformed E.
coli

1. Two stations will be set-up in class with a flask containing 500 ml of LB broth with 1.5%
agar which has been autoclaved just before class, and placed into the 65C water bath to
cool. The Laboratory Director will add ampicillin from a 50 mg/ml stock solution to
bring the solution to 200 g/ml ampicillin.

2. Label one empty bacterial culture dish on its back with your group number, meeting day,
and name (two dishes per group, one for each group member).

3. Using a 25 ml pipet and an electric pipetman, remove 21 ml of the LB-ampicillin-agar
solution from the flask and pipet it into a 100 mM bacterial culture dish. Replace the
cover, and allow the agar to solidify. This plate will be stored by the TAs in the cold box
at 4C until next period.


M-62
Experiment 8b: Culturing of E. coli

Day 16

Procedures

A. Chemical preparation of competent E. coli

1. Beginning several days before class, the Laboratory Coordinator will prepare an XL1-
blue E. coli culture for use in the laboratory by doing the following:

a. Growing colonies of XL1-blue E. coli on an LB-1.5% agar bacterial plate containing
20g/ml tetracycline (to kill any passenger bacteria that are not XL1-blue E. coli).
The XL1-blue strain of E. coli does not carry a plasmid, but has a tetracycline
resistance gene (TetR) inserted into its bacterial chromosome.

b. A bacterial loop will be used to pick one colony of XL1-blue and transferred it to 2
mls of LB liquid medium in an orange cap plastic tube. This tube will be shaken
vigorously overnight at 37C.

c. The morning of your lab period, the Laboratory Coordinator will transfer 0.5 ml of
this overnight culture into each of two 500 ml aliquots of LB medium in 3L flasks.

d. These cultures will be shaken vigorously at 37C until they grow to an A
600
of ~0.25
to 0.3. This usually takes about 1.5 to 2 hours.

To monitor growth of the cells, the lab coordinator will determine the increase in A
600

with time. These data will be made available to you in class. You should graph them
and have them in your laboratory report.

Keep your solutions with your cells ice cold from here on.

2. Transfer 40 ml of this culture to an ice-cold SS-34 centrifuge tube.

Make sure that the 0.1 M CaCl
2
solution and the 0.1 M CaCl
2
plus 15% glycerol solution
are on ice.

3. Balance your tube with that of another group be adding or taking away a small amount of
the culture solution. Place discarded culture solution in a beaker for disposal.

Centrifuge the cells for 10 min at 6,000 rpm (3300 x g) in the large centrifuges at 4C.
You should calculate the speed setting to use.

4. Pour the medium into a beaker, and discard it. Stand the tubes in an inverted position on
a paper towel briefly to remove the last traces of media.


M-63
5. Resuspend the cell pellet in 5 ml cold 0.1 M CaCl
2
.

6. Keep the cells on ice for 30 min.

7. Repeat steps 3 through 6.

8. Centrifuge the cells for 10 min at 6,000 rpm in the large centrifuges at 4C.

9. Pour the medium into a beaker, and discard it.

10. Resuspend the cell pellet in 2 ml 0.1 M CaCl
2
plus 15% glycerol solution and place on
ice. Use the serological pipette to stir and gently pipet up and down with the green
pipetman in your drawer.

These are your competent cells.

B. Transformation of plasmid DNA into the competent E. coli.

1. Transfer 200 L of competent cells from step 10 above to a 1.5 ml, sterile
microcentrifuge tube.

. Add 50 ng of plasmid DNA ([pet19b(His)LDH] to the 200 L aliquot of competent cells.

The plasmid DNA is provided at 1 g/ml. You need to determine the appropriate volume
to add to the competent cells. Check you calculation with the TAs.

3. Mix the contents of the tube by gentle swirling (!!!KEEP COLD!!!!)

4. Incubate the tube on ice for 30 min.

5. Place tube into 42C water bath for 90 seconds (Important: Just 90 sec. Not longer not
shorter).

6. Chill the tube on ice for 5 min.

7. Each group should determine their transformation efficiency by platting 20 L of
transformed cells onto one LB agar plate containing 200 g/ml ampicillin, and 80 L
onto the second plate (if you have two good plates). Use only 20 L if you only have one
good plate.

Why does this determine how many cells have taken up the plasmid in the aliquot of
cells? Because there is an ampicillin resistance gene on plasmid, which renders only
these cells resistant to the antibiotic. The untransformed cells are killed.

8. Incubate the plates in the incubator at 37C overnight.


M-64
C. Plasmid preparation

1. Add 100 L of transformed cells to 5 ml of LB media supplemented with 200 g/ml
ampicillin.

2. Incubate plates (incubator) and culture (shaker-incubator) at 37C overnight.

M-65
Experiment 8c: Plasmid Purification

Day 3

Procedures

A. Determine Transformation Efficiency

1. Count the number of bacterial colonies on the LB-agar plus ampicillin plate. If you
observe too many colonies to easily count, subdivide the plate into quarters by drawing
an X with a ruler and marking pen on the back of the plate, and count the colonies in 1
quarter of the plate.

2. Calculate transformation efficiency as colonies/g of transformed DNA.

Determine the transformation efficiency during in breaks during plasmid purification
procedure!

Notes

1. The transformation efficiency is usually about 1-5 x 10
6
/g of plasmid DNA when
using the competent cells prepared by this method.

2. Important: all steps after harvesting the cell should be done on ice (or at 4C).

Materials

E. coli containing pet19b(His)LDH plasmid to be grown in a starter culture.
Lucia Broth (LB) medium
0.1 M CaCl
2
(ice cold)
LB ampicillin agar plates
42C water bath
0.1 M CaCl
2
+15% glycerol solution, sterile

B. Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit

Notes

This protocol is designed for purification of ~20 g of plasmid DNA from 1 to 5 ml of an
overnight culture of E. coli containing pet19b(His)LDH grown in LB broth.

All protocol steps should be carried out at room temperature.


M-66
Procedure

Note: The composition of the buffers we will use here is proprietary (the company wants
to keep them secret). However, a best guess at their composition, based on the internet,
company literature, and experience, is given below.

A. Purify pet19b(His)LDH plasmid from the transformed cells

1. Centrifuge the 5 ml of cells from the Experiment 9b at full speed in the table top
centrifuges for 3 minutes at room temperature.

2. Resuspend the pelleted bacterial cells in 250 L Buffer P1 and transfer to a
microcentrifuge tube.

Buffer P1 is 5 mg/ml lysozyme; 50 mM glucose; 10 mM EDTA; 25 mM Tris-HCl pH 8.0
(Lysozyme hydrolyzes the 1,4--linkages between N-acetylmuramic acid and N-acetyl-
D-glucosamine in the peptidoglycan of the E. coli cell wall, making the cells fragile)

Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible
after resuspension of the pellet. The bacteria should be resuspended completely by
vortexing or pipetting up and down until no cell clumps remain.

3. Add 250 L Buffer P2. Mix gently but thoroughly by inverting the tube 4-6 times. Do
not vortex, as this will result in shearing of genomic DNA. If necessary, continue
inverting the tube until the solution becomes viscous and slightly clear. Do not allow the
lysis reaction to proceed for more than 5 min.

Buffer P2 is 0.2 M NaOH; 1% SDS. This osmotically and chemically disrupts the cell
membrane, denatures cellular protein and DNA, and hydrolyzes RNA.

4. Add 350 L Buffer N3, and mix immediately and thoroughly by inverting the tube 4-6
times.

To avoid localized precipitation, mix the solution thoroughly immediately after addition
of Buffer N3. The solution should become cloudy.

Buffer N3 is Guanidine hydrochloride; acetic acid; isopropanol; potassium acetate; and
acetic acid, pH 4.8. This solution neutralizes the NaOH from the previous step,
renaturing the plasmid DNA, but leaving the more complex cellular DNA denatured and
insoluble, and also solubilizes the protein. Guanidine hydrochloride is a strong
chaotrope.

5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A
compact white pellet will form.

6. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.

M-67

7. Centrifuge for 30-60 s. Discard the flow-through.

8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60s.

Buffer PE contains ethanol and is low ionic strength (salt concentration).

9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash
buffer.

Important: Residual wash buffer will not be completely removed unless the flow-through
is discarded before this additional centrifugation. Residual ethanol from Buffer PE may
inhibit subsequent enzymatic reactions.

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute
pet19b(His)LDH plasmid DNA, add 50 L Buffer EB (10 mM Tris-Cl, pH 8.5) or water
to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

B. Determine the concentration of the DNA.

1. Turn on the UV lamp of the spectrophotometers, wait 20 minutes for the lamp to warm
up.

2. Dilute 10 L of the DNA from step 10 above into 90 L of deionized (d.i.) water in an
Eppendorf tube. Vortex briefly, and centrifuge in the Eppendorf centrifuge briefly to
return the solution to the bottom of the tube.

3. Determine the A
260
and A
280
of the diluted DNA solution. Blank against d.i. water before
and between readings.

4. Determine the concentration and purity of the DNA.

To calculate the concentration of DNA, use the formula:

1 A
260
unit = 50 g DNA/ml

An A
260
/A
280
ratio > 1.8 suggests little protein contamination in a sample.

M-68
Experiment 9: Restriction analysis of DNA

a. Set up the restriction enzyme reactions

1. Each member of the team will perform a restriction enzyme digestion of the isolated
pet19b(His)LDH plasmid DNA. Each member will set up two reactions one with XhoI
restriction enzyme, the other with NdeI and XhoI restriction enzyme (see Figure 2).

Set up the restriction enzyme digest as shown below (listed in order of addition):

Reaction 1
x L of plasmid DNA (minimum: 0.5 g total)
2 L 10X Reaction Buffer 2
2 L of 10xBSA (1 mg/ml)
Sufficient d.i. H
2
O to adjust the total reaction volume to 20 L
2 L Xho1 (1 unit/L)

Reaction 2
Sufficient d.i. H
2
2 L Xho1 (1 units/L)
2 L Nde1 (1 units/L)

Reaction 3
Add a sufficient amount of d.i. H
2

2. Briefly vortex the samples and pool the reagents at the bottom of the tube by a brief (10
sec) spin in the microcentrifuge.

3. Incubate reaction tubes 30 min at 37C (in a water bath or incubator).

4. Heat inactivate your samples by incubating them at 65C for 20 minutes

5. Add 1/10 volume (2 L) of 10 x DNA loading dye. Mix well by gently tapping tube.

The laboratory coordinator will freeze your samples for next laboratory period.


M-69
b. DNA agarose gel electrophoresis: Gel preparation

1. Thaw your reactions by placing them at room temperature.

2. You will need 1000 ml of 1 x TBE buffer for the gel apparatus. You will need an
additional 300 ml 1 x TBE buffer to prepare the gel.

3. Tape ends of gel holder and insert comb. Make sure that there is a paper width of space
between the comb and the bottom of the gel tray and that the comb is level. Make sure
the gel holder is also level.

If the comb touches the bottom of the gel tray, when you load the sample on the gel, it
will spread out under the gel, along the bottom of the tray.

4. Prepare agarose

1 % Agarose (Seakem LE) = 3.2 g
1 x TBE = 300 ml

Combine the agarose and TBE in 500 ml flask. Cover top of flask with plastic wrap and
punch a small hole in the top. Heat in the microwave for 30 seconds/time until dissolved.
Swirl after each heating. If almost dissolved, heat for shorter periods of time.

Caution: Agarose can develop superheated spots and can explode when swirled. Use
hot gloves and heat just to boiling.

5. Add 30 L ethidium bromide stock solution (10 mg/ml)

Caution: wear gloves. This solution is mutagenic.

6. Cool a little first to prevent warping of the holder. Pour the agarose solution into the gel
holder.

7. Cool for 30 minutes before use.

8. Remove tape and insert the gel into the electrophoresis chamber.

9. Record your well numbers in the agarose gel.

10. Load your samples and the DNA marker in separate wells of the gel. Load the samples in
the wells using a P20 micropipette. Change tips between samples. Place the tip of the
pipet containing the sample to be loaded under the buffer, just at the opening of the well.
The loading buffer contains glycerol, which increases the density of the sample to be
loaded. The sample will settle in the well because it is more dense than the buffer. Be
careful not to poke a hole in the bottom of the well with the pipette tip.


M-70
11. Place the cover on the gel electrophoresis unit and plug the leads into the power pack.
Make sure the positive lead runs from the bottom of the gel (DNA is negatively charged
and will run to the positive pole).

Caution: electrical hazard. Always turn off the power pack and unplug the leads .
before opening the electrophoresis unit. Do not pull on the wires. This level of
electricity can kill.

12. Turn on the power and run the gel at 150 V for 30-45 min or until the tracking dye is 2/3
to 3/4 of the distance to the end of the gel.

13. Turn off the power.

14. Unplug the leads by pulling on the plastic clips. Do not pull on the wires.

15. Remove the top of the unit and take out the gel and gel holder (caution, the gel can slide
off the holder.

Caution: wear gloves. This solution is mutagenic.

16. Stain for 15 minutes. Remove from stain using slotted scoop and place on clear plastic
wrap. Use the plastic wrap to move the gel to a transilluminator.

Caution: A transilluminator produces UV radiation in the 254 nm range. This
wavelength can cause eye damage (short term = burns, long term cataracts and skin
cancers). Always wear UV protection goggles.

17. Draw or photograph your gel according to instructions.

c. Analysis of Results

1. Determine size (DNA length in bp) of digested and undigested plasmid DNA. Use semi-
log paper to draw a curve comparing DNA length with distance migrated. First establish
standard (marker) curve by plotting the Rf of marker DNA against size (bp) of DNA.
Calculate Rf of plasmid and determine size of plasmid using the standard curve.

2. Compare size of plasmid DNA digested with NdeI and XhoI. Determine size of DNA.
How do you interpret the results?

3. Draw a map of the provided plasmid.

M-71
Materials

5X TBE electrophoresis buffer (Tris Borate EDTA)
108 g Tris base
55 g Boric Acid
40 ml of 0.5M EDTA, pH 8.0
5 mg Ethidium Bromide = 2.5 ml of 10 mg/ml stock (optional)

10 X Loading dye
7.5 g Ficol 400
0.125 g bromophenol blue
Bring to 50 mls with H20

DNA Molecular Weight Markers: 1 kb DNA Ladder

Ethidium Bromide (EtBr) Staining Solution (10 mg/ml)

Caution: Ethidium Bromide is a powerful mutagen. Always use gloves when handling
anything that has been exposed to ethidium bromide. Dispose of ethidium bromide to
glass containers marked for that purpose. Sunlight will beak down ethidium bromide and
may be used to decontaminate stocks. For use, keep ethidium bromide stocks out of the
light.

References

Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. (1970)
53:159-162.


M-72
Appendix

Appendix Lab 1. Purification of LDH from chicken

Protease Inhibitors PMSF and EDTA

M-73

I. Protein Source
Methods for disrupting cells
gentle
Moderately vigorous
vigorous
vigorous

M-74
Separation of soluble and insoluble molecules (e.g.
proteins) from insoluble material (cell debris,
insoluble proteins) by centrifugation

M-75
Centrifugation

Consider particle of mass mp spinning in a sector-
shaped centrifuge cell

Particle experiences 3 forces:

(Force required to
push displaced
solvent aside)

M-76

Therefore:

M-77


M-78
Conversion RPM to RCF


M-79
Appendix Lab 2

Immobilized Metalion Affinity-Chromatography
(His-Tag = multiple >6 Histidine residues)

NTA chelating group (nitrilotriacetic acid) with ligated Ni2+
ion (note presence of two empty coordination positions
here occupied by H2O molecules). (Other chelating groups
may Ligate Co
2+
instead of Ni
2+
)

Two His residues of His-tag attached to a complexed Nickel
ion on Ni-beads (protein can be eluted with imidazole or
histidine)
Ni-NTA
spacer
Matrix
(resin)

M-80
Purification of His-tagged proteins under native and
denaturing conditions


M-81
Appendix Experiments 4, 5, and 6

This appendix applies especially to:

Experiment 5: Yield and Specific Activity of LDH
Experiment 6: KM and kcat of uninhibited LDH
Experiment 7: KM and kcat of inhibited LDH

Kinetic studies of enzyme-catalyzed reactions provide information about an enzyme's
mechanisms specificity, and other parameters. The -practical applications of enzyme kinetics are
widespread. Kinetic assays in vitro are widely used in clinical laboratories because an abnormal
enzymatic reaction rate can provide a clue to a pathological condition. Kinetic assays in vivo
provide information about the rates of individual enzymes and entire metabolic pathways.
Studying inhibitors of enzymes helps us to understand an enzyme's mechanism. By using
inhibitors that are structural analogs of the substrate, insight into the active site of an enzyme can
be obtained. Once the active site's chemistry has been determined, it is sometimes possible to
design inhibitors with therapeutic value.

General Principles of Enzyme Kinetics

Enzymes are catalysts in that they increase the rate, or velocity, of chemical reactions,
without themselves being changed in the process. Catalysts do not change the thermodynamic
equilibrium between reactants or products, they only expedite the attainment of thermodynamic
equilibrium. For chemical reactions to occur, the reactants must collide in the correct orientation
and with sufficient energy to overcome the Activation Energy that is associated with the
reaction. By correctly orienting the reactants and by lowering the activation energy, enzymes
speed up reaction rates by factors of 10
3
to 10
16
compared to the corresponding uncatalyzed
reactions. Most enzymes are also highly specific, capable of discriminating between reactants
(known as "substrates" in enzymological terminology) that are nearly identical, such as
stereoisomers. The products of enzymatically-catalyzed reactions are formed in nearly 100%
yield, and are free of the side-products that are typical of uncatalyzed reactions.
The magnitude of the activation energy determines the rate of reaction. Transition state
theory describes how enzymes lower the activation energy. In the transition state the enzyme-
substrate complex is in its most energetically unfavorable configuration, with chemical bonds in
the process of being broken or formed. The reaction rate, k, is related to the activation energy,
G
, by:

where R is the gas constant and T is the absolute temperature in degrees Kelvin. Recall that

where H
is the enthalpy of activation and S

is the entropy of activation.

M-82
Energetics of the Initial Interaction between the Substrate and Enzyme

Entropy of Activation: When two reactants collide to form a single product the translational
and rotational entropy of one of the reactants is lost. This entropy loss is considerable, yielding a
negative S for the reaction, which increases the magnitude of G
binding
. (Because there is a
negative sign in front of TS in the equation G=H - TS, G becomes larger as S becomes
more negative, i.e., less random motion). However, if the translational and rotational motion,
termed degrees of freedom, of the reactants are already restricted because an enzyme is
holding the reactants rigidly in place, there is little entropy loss when the actual reaction takes
place. Enzymes, therefore, partly minimize G
by minimizing S
in the rate-limiting step. This

removes most of the entropic unfavorability of the reaction during the process of binding the
substrate. Thus, most of the entropic unfavorability of the chemical reaction is incurred when the
substrate binds to the enzyme, rather than in the chemical reaction itself.

Enthalpy of Activation: Although the formation of an enzyme-substrate complex is highly
unfavorable entropically, this is overcome by a highly favorable change in enthalpy (H
binding
)
due to the formation of new hydrogen bonds, electrostatic interactions, and van der Waals
interactions between the substrate and enzyme. But, the entropic unfavorability of binding
substrate is important in resisting the big decrease in enthalpy that occurs as a result of these
interactions, because it weakens the binding of the substrate to the enzyme somewhat. Maximal
rates of enzymatic reactions are achieved when the binding of the substrate to the enzyme is
mildly unfavorable. We will discuss the reason for this more below.

The transition state of the enzyme-substrate complex: Overall, enzymes make the G
of
a reaction smaller by making favorable contributions to H
in the transition state, through

hydrogen bonds, electrostatic interactions, and van der Waals attractions, and by minimizing S

in the transition state (e.g., random motion). Enzymes minimize S
in the transition state by

incurring an unfavorable S while binding the substrate. The entropic unfavorability of binding
substrate can be wholly or partly overcome by hydrogen bonds, electrostatic interactions, and
van der Waals contacts with the substrate. Maximal reaction rates are achieved when the initial
binding reaction is mildly unfavorable (see figure below).


M-83

Figure explanation: Reaction profile for an enzyme-catalyzed reaction (arrow labeled catalyzed)
compared to a reaction profile for an uncatalyzed reaction (arrow labeled uncatalyzed). The
enzyme-substrate complex (ES) has a lower energy than the isolated enzyme (E) and substrate
(S) separately (E + S). The transition state of the enzyme-catalyzed reaction (ES
) also has lower

energy than the transition state of the uncatalyzed reaction (S
). The decrease in energy (G
) of
the transition state for an enzyme-catalyzed reaction exceeds the decrease in energy (G
b
) that
occurs upon binding of the enzyme to substrate. Formation of the enzyme-substrate complex
thus decreases the activation energy (G
a
) of the reaction (from G
a-uncat
to G
a-cat
) and thereby
increases the reaction rate. The difference in energy (G) between the reactants (S) and products
(P) is the same for an enzyme-catalyzed or uncatalyzed reaction. Thus, the enzyme does not
alter the equilibrium constant for the reaction. (Illustrator: Mike Webb; taken from Biochemistry
by J.D. Rowan, 1989)
Course of the reaction
Change in the
transition
state energy
Change in energy
upon formation of
enzyme-substrate
complex
Difference in energy
between reactants
and products
Ener
gy
S
G
a-cat
G
b
ES
E+P
G
ES
E+S
G
a-uncat
EP

M-84
Formal Consideration of Overall Enzyme Kinetics

The catalytic mechanism and substrate specificity of an enzyme can be determined by
studying the kinetics of the enzyme-catalyzed reaction as a function of substrate concentrations.
Information concerning the structure of the catalytic site can be determined.by studying how
inhibitors affect these kinetics.

For a simple unireactant system, the following equilibria can be written:

where E, S, ES and P refer to the concentrations of free enzyme, free substrate, enzyme-substrate
complex, and product, respectively.

The rate of product formation will gradually decrease as the substrate is depleted and product
is formed, as illustrated below.

Figure legend: Progress curve for an enzymatic reaction. A single
substrate is converted to product (P). The progress curve for an
enzymatic reaction is linear (near t = 0), when very little substrate has
been converted to product. The slope of the curve at time zero is the
initial rate of the reaction. (Illustrator: Lisa Shoemaker.)

However, if one considers the reaction before there is substantial depiction of substrate or
accumulation of product, then one can ignore k
-2
and write:

If the concentration of substrate is much greater than the concentration of enzyme, then very
soon after mixing E with S, the concentration of the ES complex will remain nearly constant
with time. Under these steady-state conditions, the rate of formation of the ES complex is
approximately equal to the rate of its decomposition. See the figure below.

Slope at t = 0

M-85

Assuming that steady-state conditions apply, one can write the following differential expression
for the rate of change of [ES] with time, where the initial concentration of substrate is [S]
o
:

Rearranging, making several assumptions, and defining the initial enzymatically catalyzed rate
v
o
as k
2
[ES], the equation rearranges to:

Where the K
M
is the Michaelis constant, which is defined as:


M-86
This is the expression that relates the initial rate of product formation or substrate consumption,
V
o
(also known as the initial velocity), to the initial substrate concentration, [S]
o
, for an enzyme
catalyzed reaction that follows Michaelis-Menten kinetics. Many more complicated enzyme
mechanisms also produce kinetics that follow this same expression. Consequently, the
Michaelis-Menten expression is written more generally as:

with

An examination of the limits of the Michaelis-Menten expression provides some insights.

I. If [S]
o
>> K
M
, then v
o
= k
cat
E
o
= V
max
(See page 98 for a discussion of k
cat
)

Under these conditions, the enzymatic rate is the maximum that can be achieved for the
concentration of enzyme employed.

II. If [S]
o
<< K
M
, then

Under these conditions, the initial enzymatic rate is first order in substrate concentration, and
the initial velocity is equal to

M-87
It should be remembered that, because the Michaelis-Menten expression was derived with
the assumption that substrate depletion and product formation were negligible, one must take
care to measure the true initial rates of a reaction when estimating the kinetic parameters of any
enzyme.

III. Interpretation of the Michaelis-Menten parameters.

1. When v
o
= (1/2)V
max
, K
M
= [S]
o

2. K
M
can be considered to be an apparent dissociation constant, K
D
, for the substrate.
However, it is not a true K
D
unless k
2
(k
cat
) << k
-1

3. K
M
is a constant for a particular enzyme. A determination of K
M
permits comparison of the
same enzyme isolated from different organisms, or from different tissues of the same
organism. In this manner one can identify different enzymes that catalyze the same reaction.

4. The physiological substrate concentrations ([S]
o
) are known for a few enzymes (see Table).

M-88
For most of these enzymes K
M
is 1- to 100-fold larger than [S]
o
. There should be little
evolutionary pressure for an enzyme to evolve a K
M
that was substantially smaller than the [S]
o
,
since even a 100-fold decrease in the ratio of K
M
to [S]
o
would increase v
o
by less than a factor
of 2. On the other hand, it is catalytically advantageous for K
M
to be somewhat larger than [S]
o

(this point will be discussed below). It also makes metabolic sense to have K
M
> [S]
o
, since
under these conditions the reaction rate is directly proportional to [S]
o
, and is therefore
maximally sensitive to fluctuations in [S]
o
. However, if K
M
was significantly more than 100-fold
larger than [S]
o
, v
o
would always be far less than V
max
, and most of the catalytic potential of the
enzyme would be wasted.

5. The activity of some enzymes is regulated by ligand-induced conformational changes that
may significantly alter K
M
. An unreasonably large K
M
may indicate that the enzyme is activated
by another component in vivo.

6. If an enzymes K
M
and k
cat
are known, its concentration can be determined by measuring v
o

under conditions where [S]
o
>> K
M
.

The Catalytic Constant k
cat
.

1. In the simple unireactant mechanism considered above, k
cat
is simply the first order rate
constant for converting the ES complex to EP. In more complicated reaction mechanisms, k
cat
is
a function of all the microscopic rate constants involved in the reaction mechanism. k
cat

represents the overall catalytic rate of an enzyme. It represents the number of substrate molecules
converted to product per second. It is also referred to as the turnover number of the enzyme.
Turnover numbers of 10
3
sec
-1
are typical.


M-89
One international unit (1 U) of enzyme activity is the amount that catalyzes the formation of 1
mole of product per minute under defined conditions.

k
cat
/K
M
The specificity constant.

Under conditions where [S]
o
<< K
M
, the Michaelis-Menten equation becomes:

Under these conditions, only a small fraction of the enzyme has substrate bound to it, so one can
approximate E
o
= [E]
total
[E]
free
one has the expression:

Hence, at low substrate concentrations (when [S]
o
<< K
M
), the ratio k
cat
/K
M
becomes an apparent
second order rate constant. This ratio indicates the catalytic efficiency of an enzyme for a given
substrate. Its value ranges from less than 1, to greater than 10
8
sec
-1
M
-1
. If one considers the
simple unireactant system described above, k
cat
= k
2
, and the ratio k
cat
/K
M
becomes:

If k
2
>> k
-1
, then

In other words, if k
2
(or k
cat
) is very large, then the binding of substrate becomes the rate limiting
step in the enzyme mechanism. The binding of substrate can be no faster than the time it takes
for a substrate molecule to collide with the enzymes active site. From col1ision theory, the
maximal rate that a small molecule can collide with a particular part of a larger molecule is 10
9
-
10
11
sec
-l
M
-l
. No enzyme can have a k
cat
/K
M
ratio higher than this value. A number of enzymes
have k
cat
/K
M
ratios as high as 3 x 10
8
sec
-1
M
-1
. These enzymes are said to have k
cat
/K
M
ratios that
are close to the diffusion controlled limit, and are considered to have catalytic efficiencies that
are nearly perfect. These enzymes must also have K
M
, values that are significantly higher than
the physiological concentration of substrate (see below).


M-90
The ratio k
cat
/K
M
is also useful for describing the specificity of an enzyme for its substrate. The
ratio will be largest for an enzymes prefer red substrate.

IV. The Maximal Efficiency of an Enzyme.

An enzyme achieves maximal efficiency when the ratio of k
cat
/K
M
is maximized, and when
K
M
is at least 10-fold greater than the physiological concentration of substrate ([S]
o
). This point
is best explained by transition state theory. Enzymes catalyze reactions by stabilizing the
substrate configuration in the transition state, thereby decreasing G
. However, if an enzyme
has K
M
< [S]
o
, (so that substrate binding is strong) then the ES complex is stabilized with respect
to the uncomplexed enzyme plus substrate (E + S). Stabilizing the ES complex increases the
G
, however. Ideally one would like to stabilize the transition state, while concurrently
destabilizing the ES complex with respect to E + S. Under these circumstances some of the G

of the transition state is overcome by substrate binding, so that the G
of the catalytic reaction

is decreased. To destabilize the ES complex with respect to E plus S, one wants K
M
to be at least
10-fold greater than G
, so that substrate binding is weak.


M-91

Reactions involving stickase

V. Enzyme Assays

The course of enzyme catalyzed reactions is frequently monitored spectrophotometrically.
Occasionally the substrate or product has a characteristic absorption band. By monitoring this
absorption band over time, the rate of change of the concentration of the substrate (or product)
can be measured. Usually, however, neither the physiological substrate nor the product have

M-92
readily accessible absorption bands. However, the kinetic parameters of many enzymes can be
determined with artifica1 substrates. Such substrates (or the products that are derived from them
following enzyme action) typically have characteristic absorption bands in the visible.

In developing the Michaelis-Menten rate equation, we assumed that depletion of substrate
and accumulation of product were negligible during the course of the assay. This assumption was
made so that we could ignore the slowing of the reaction as the substrate concentration was
depleted, and so that we could ignore possible inhibition of the enzyme by product. With this
assumption, [S]
o
is constant during the course of the assay. However, this assumption is true only
during the first moments of an assay.

Because the reaction rate diminishes with time, and because of the assumptions inherent in
the derivation of the Michaelis-Menten rate equation, extraction of the Michaelis-Menten kinetic
parameters requires measuring the initial rate of the enzyme reaction immediately. after substrate
and enzyme are mixed..


M-93
Ideally, the reaction can be monitored continuously by spectrophotometry. In many cases this
is not possible, however, either because the appropriate spectrophotometric equipment is not
always available to permit continuous monitoring over time, or because neither substrate nor
product have readily accessible absorption bands. Even when artificial substrates are employed,
it may be necessary to treat the enzyme-substrate-product mixture after the reaction to convert
the substrate or product to a compound that has an appropriate absorption band.

To extract the kinetic parameters that define an enzymatic reaction, one must measure v
o
at
many different initial substrate concentrations. Normally one choses numerous [S]
o
values
between 0.5 K
M
and 10 K
M
. Higher substrate concentrations produce little additional data. For
example, doubling [S]
o
from 5 K
M
to 10 K
M
increases v
o
by less than 10%, from 0.833 V
max
to
0.909 V
max
. In addition, many substrates are quite expensive; [S]
o
values higher than 10 K
M
may
consume considerable quantities of substrate.

If continuous monitoring of the enzymatic reaction is not possible, a fixed time assay might
be employed. In a fixed time assay, the enzyme-catalyzed reaction is allowed to proceed for a
fixed time interval, then the reaction is rapidly stopped by modifying or denaturing the enzyme.
The amount of product produced (or substrate remaining) is then determined. Obviously, because
an interval of time is employed, a true initial rate cannot be measured. However, it is possible to
extend the linear region of the reaction progress curve by conducting the assay under
conditions where [S]
o
> > E
o
(subject to the provision that, for estimating kinetic parameters, one
should use [S]
o
values within the range of 0.5 K
M
< [S]
o
< 10 K
M
). Generally, the progress curve
will be near-linear only while > 95% of the substrate remains.

To carry out a fixed time assay, one must chose appropriate values for E
o
and [S]
o
. Typically,
one choses an appropriate enzyme dilution such that the progress curve is linear (within
experimental uncertainty) throughout the desired time interval at all substrate concentrations that
are to be employed (0.5 K
M
< [S]
o
< 10 K
M
).


M-94


M-95
Example:

1. The stock solution of hog muscle LDR has a concentration of 10.0 mg protein/mL.

2. A freshly-prepared 500-fold dilution of this stock solution is used in the activity assay
{measurement of the initial rate of conversion of (lactate + NAD
+
) to (pyruvate +NADH).

3. An aliquot of 10 L of the 500-fold diluted LDH is added to the assay mix.

4. The assay volume 1.2 mL

From the data on the graph shown on the next page, one can calculate that:

1. The initial rate of product formation in the cuvette is:

vo = 0.0341 moles/min = 0.0341 units.

2. The activity of the 500-fold diluted enzyme is 3.41 units/mL.

3. The activity of the UNDILUTED enzyme stock is 1700 units/mL.

4. The Specific Activity of the enzyme stock is 170 units/mg.

5. We know that V
max
= k
cat
E
o
. If we assume that v
o
is approximately 85% of V
max
under the
conditions of our assays, then we can calculate:

Initial activity in assay mix
Enzyme concentration in
assay mix
dilution
volume
used in
assay
MW of LDH

M-96


M-97

Examples from Segal, I.H. (19 76) Biochemical Calculation


M-98

M-99

M-100


M-101

M-102

VI. Extracting K
M
and V
max
from Kinetic Data.

To estimate K
M
and V
max
from kinetic data, one measures the initial rates of product
formation at different initial concentrations of substrate. Because the Michaelis-Menten rate
equation was derived by assuming that depletion of substrate (and formation of product) is
negligible during the assay, one must be careful to measure only the initial rate of product
formation.

It is not possible to accurately estimate V
max
or K
M
from plots of v
o
versus [S]
o
because v
o

only approaches V
max
asymptotically as [S]
o
. Therefore, it is common practice to rearrange
the Michaelis-Menten expression so that data can be plotted in a form that yields a straight line.
Several rearrangements are in use.

A. The most common graphical rearrangement of the Michaelis-Menten rate equation is the
double-reciprocal, or Lineweaver-Burk plot.

Therefore:


M-103

The Lineweaver-Burk plot has numerous disadvantages.

1. Because reciprocals of the data are computed, small experimental errors can lead to very
large uncertainties in the plotted values.

2. The least reliable data (those taken at the lowest values of [S]
o
) are invariably given the
most weight by eye.

3. The most reliable data (those taken at higher values of [S]
o
) are compressed to a small
area on the plot.

The only reliable method for extracting K
M
and V
max
values from a Lineweaver-Burk plot is
to calculate a weighted least-squares fit to the data points. A simple least-squares fit that does not
take into account the relative uncertainties of each point is no better than fitting a line by eye.
The use or the Lineweaver-Burk plot for extracting kinetic parameters is discouraged today.
Superior methods exist, as discussed below.


M-104

B. v
o
versus v
o
/[S]
o
, or Eadie-Hofstee plot.

This plot is an improvement over-the Lineweaver-Burt plot, but still suffers from distortions
in the uncertainties of the plotted data, and from compression of data. Precise values for K
M
and
V
max
must again be extracted from a weighted least-squares fit of the plotted data points.
Furthermore, because the experimental uncertainty is present on both axes of this plot, the
regression analysis is more complicated. Standard analyses assume that all of the uncertainty is
in the dependent variable.

Therefore,

c. [S]
o
/v
o
versus [S]
o
, or Hanes-Woolf plot.

This is a better graphical representation of the Michaelis-Menten equation. The plotted data
are spread out better, and the uncertainties are not as distorted.


M-105

D. The direct linear plot.

The newest (and best) method introduced for extracting K
M
and V
max
values is the direct
linear plot of Cornish-Bowden and Eisenthal. For each pair of experimentally determined v
o
and
[S]
o
values, a straight line is drawn with intercept -[S]
o
on the x axis, and v
o
on the y axis.
All lines should intersect at a single point that determines the K
M
and V
max
values.

Therefore,


M-106


M-107
The direct linear plot has several distinct advantages over all the others mentioned earlier.

1. No calculations are required. Each pair of data points can be plotted as it is obtained.

2. Outlying data points can be immediately spotted. New values can be experimentally
determined immediately to determine whether the outlying value is the result of poor
technique, or the result of the enzyme mechanism not following Michaelis-Menten
kinetics.

3. The extracted K
M
and V
max
values are median values, rather than average values, and
therefore they are much less affected by a few bad values. The uncertainties of the K
M

and V
max
values can be estimated very readily.

In the figures below, the same data is presented with a Lineweaver-Burk plot (left) and a
direct linear plot (right). Note how the erroneous v
o
values obtained at the lowest [S]
o
values
stand out in the direct linear plot, but may lead to erroneous estimates of K
M
and V
max
with the
Lineweaver-Burk plot.


M-108
VII. Enzyme Inhibition.

Any substance that diminishes the rate of an enzymatically-catalyzed reaction is an inhibitor.
Enzyme inhibition is a major facet of metabolic regulation. Studying inhibition of an enzyme in
vitro can yield information about the mechanism of the enzyme, the enzyme's preference for
substrates, the structure of the enzyme's active site, and the structure of the substrate-enzyme
transition state complex. Inhibitors can be drugs, antibiotics, poisons, toxins, or the products of
enzyme-catalyzed reactions.

Competitive Inhibition:

Competitive inhibitors reversibly bind to an enzyme and prevent the substrate from binding.
Sometimes the inhibitor and substrate have similar structures and compete for the same binding
site. Often, however, the inhibitor and substrate have very different structures, and bind to
different sites simultaneously. Alternatively, binding of the inhibitor may induce a

M-109
conformational change in the enzyme that prevents the substrate from binding. Frequently, an
enzyme near the beginning of a metabolic pathway is inhibited by an end product of that
pathway. This phenomenon is known as "feedback inhibition. Feedback inhibition frequently
involves inhibitor-induced conformational changes.

Competitive inhibition can be overcome by adding high concentrations of substrate. V
max
can
still be achieved, but the apparent affinity of the substrate for the enzyme will be diminished, and
thus, K
M
will increase.

K
m, apparent

As [S]o , v
o
v
max
, as before.


M-110

M-111
Competitive inhibition is a special case of inhibition. A more general treatment includes the
possibility that the inhibitor binds to the ES complex:

In purely competitive inhibition, one assumes that K
I(ES)
is essentially infinite (i.e., the inhibitor
cannot bind to the ES complex). In many cases, however, the inhibitor can bind either to the free
enzyme (E) or to the enzyme-substrate complex (ES). Further, it can bind and bind to the free
enzyme and the enzyme-substrate complex with different affinities. In this case one observes
Mixed Inhibition. The reason for referring to this general case as "mixed" should become
apparent after two limiting cases are considered. (Neither limiting case is very common).

A. Limiting case where the inhibitor binds only to the ES complex: Uncompetitive
Inhibition.

In this case, the inhibitor does not bind to the free enzyme: one assumes that K
I(E)
is essentially
infinite. Because the inhibitor diverts some of the ES complex to the inactive ESI complex, the
inhibitor decreases V
max
at all substrate concentrations. The diversion of some of the ES complex
effectively shifts the E + S ES equilibrium to the right, increasing the apparent affinity of the
substrate for enzyme, thereby decreasing K
M
.

M-112

So, V
max, apparent
= and K
m, apparent
=

So, when the inhibitor can bind only to the ES complex, both V
max
and K
M
are decreased by
identical factors. This limiting case of inhibition is referred to as uncompetitive inhibition. It
is rare that an inhibitor that binds to the ES complex will have no affinity for the isolated
enzyme. So, examples of uncompetitive inhibition are not very common. Uncompetitive
inhibition appears to occur only in some forms of product inhibition. It is much more common
for an inhibitor to bind to both the free enzyme and to the enzyme-substrate complex.


M-113
B. Limiting case where K
I(E)
= K
I(ES)
: non-competitive inhibition.

In this limiting case, the inhibitor binds to the free enzyme and to the enzyme-substrate complex
with equal affinity. Under these conditions the E + S ES equilibrium will be unaffected
because the concentrations of both E and ES are diminished equally by the inhibitor. Although
V
max
will be decreased because some of the ES complex is diverted to the inactive ESI complex,
K
M
will be unaffected. Therefore,

Therefore,
and K
M uninhibited
= K
M inhibited


M-114
Under the conditions where the inhibitor binds to the enzyme and to the ES complex with equal
affinity, V
max
is decreased, but K
M
is unaffected. This limiting case of inhibition is referred to as
NONCOMPETITIVE inhibition. For these conditions to apply, the inhibitor must interfere
with the catalytic mechanism, but have no effect on the binding of substrate. Examples of such
inhibitors are protons and heavy-metal ions. Other examples are exceedingly rare.

MIXED INHIBITION
In most cases of inhibition, the inhibitor binds to both the free enzyme and to the ES complex
with different affinities. The Michaelis-Menten equation becomes more complex in this

K
M, apparent

In mixed inhibition, V
max
is decreased by the same extent as in the limiting cases of
uncompetitive and noncompetitive inhibition, but K
M
is either increased or decreased, depending
on the relative magnitudes of K
I(E)
and K
I(ES)
. Most examples of product inhibition correspond to
mixed inhibition.

VIII. Graphical Methods for Plotting the Results of Enzyme Inhibition.

Because inhibitors change K
M
and
V
max
values, the standard Lineweaver-
Burk, Eadie-Hofstee, Hanes-Woolf, and
Cornish-Bowden direct-linear plots are all
changed by inhibition.

Whether inhibition is mixed or purely
competitive can be readily determined, or
displayed, with the use of an appropriate
plot.


M-115

Note the changing position of the intersecting lines in the direct linear plot. For purely
competitive inhibition, the intersection point shifts directly to the right because V
max
is
unchanged. For noncompetitive inhibition, the intersection point shifts direct1y downward
because K
M
is unchanged. For uncompetitive imbibition, the intersection point moves toward the
origin because both K
M
and V
max
change to the same extent. For the more common case of
mixed inhibition, the intersection point moves both downward and either to the right or to the
left because V
max
is decreased and K
M
is either increased or decreased.

Also note the position of the intersecting lines in the Lineweaver-Burk plot. For purely
competitive inhibition, the intersection point lies on the 1/v
o
axis because V
max
is unchanged. For
noncompetitive inhibition, the intersection point lies on the l/ [S]
o
axis because K
M
is unchanged.
For uncompetitive inhibition, the lines fail to intersect because both K
M
and V
max
change to the
same extent. In the more common case of mixed inhibition, the intersection point lies on neither
axis because both K
M
and V
max
are changed.


M-116

Key Concepts - Review


M-117


M-118


M-119


M-120


M-121
References
1. Rawn, J.D. (1989) Biochemistry, Carolina Biological Supply Company, Bur1ington, pp. 149-
156, 166-186.

2. Wood, W.B., Wilson, J.H., Benbow, R.M. and Hood, L.B. (1981) Biochemistry: A Problems
Approach, 2nd Edition, The Benjamin/Cummings Publishing Company, Menlo Park, pp.144-
151.

3. Fersht, A. (1985) Enzyme Structure and Metabolism, 2nd Edition, W.H. Freeman and
Company, New York, pp. 98-109, 311-317, 324-331.

4. Cark, J.M., Jr., and Switzer, R.L (1977) Experimental Biochemistry, 2nd Edition, W.H.
Freeman and Company, New York, pp. 82-84, 105108.


M-122
BCH 102 Laboratory Report
Experiment #1: Isolation and Characterization of Lactate Dehydrogenase form chicken

Student Name (Last, First): ,
.
Date Received: .

Points received/Points Possible: 90 Total Points (9 pts/day deductions up to a total of 90
points)

Title Page: / 5 pts

Objective: / 5 pts General overview of experiments and their purpose
/ 2.5 pts Purification
/ 2.5 pts Bradford Assay

Procedures
A. Expt. 1a-c Purification: / 10 pts
/ 2.5 pts Homogenization in presence of PMSF and 2-ME
/ 2.5 pts AmSO
4
precipitation
/ 2.5 pts Resuspension and Desalting
/ 2.5 pts Cibacron Blue Chromatography
B. Expt. 1d Bradford Assay Protein Concentration: / 5 pts
/ 2.5 pts Standard curve
/ 2.5 pts Dilutions

Results
A. Purification: / 25 pts
/ 5 pts Observations of purification steps for chicken LDH
/ 5 pts Plot A
280
, protein concentration vs. fraction # for chicken LDH
/ 5 pts Bradford graph, data, calculations, and standard curve to
estimate protein concentration for bacterial LDH
/ 10 pts purification table and calculations

Discussion: 40 pts


M-123
BCH102LaboratoryReport
Experiment#2:IsolationandCharacterizationofbacterialLactateDehydrogenaseusingmetalion
chromatography
StudentName(Last,First):
DateReceived:
Pointsreceived/PointsPossible:
TitlePage:____/5pts
Objective:____/5ptsGeneraloverviewofexperimentsandtheirpurpose
____/2.5ptsMetalionchromatography
____/2.5ptsBradfordAssay
Procedures
A.Expt.2acPurificationwithNickelaffinitychromatography:____/10pts
____/2.5ptsExpression(graph)
____/2.5ptsHomogenization
____/5ptsMetalionchromatography(detaileddescription)
C.Expt.2dBradfordAssayProteinConcentration:____/5pts
____/2.5ptsStandardcurve
____/2.5ptsDilutions
Results
A.Purification:25pts
____/5ptsObservationsofpurificationstepsforbacterialHistaggedLDH
____/5ptsPlotA280,activityandproteinconc.vs.fraction#forchickenLDH
____/5ptsBradfordgraph,data,calculations,andstandardcurvetoestimateproteinconcentrationfor
bacterialLDH
____/ 10 pts purification table and calculations
Discussion:40pts


M-124
BCH 102 Laboratory Report
Experiment #1-7: Isolation and Characterization of Lactate Dehydrogenase

Student Name (Last, First): , .
Date Received: .

/ 200 Total Points (20 pts/day deductions)

Points received/Points Possible:

Title Page: / 5 pts
Objective: / 10 pts General overview of experiments and their purpose

/ 2.5 pts SDS-PAGE & tertiary structure
______/ 2.5 pts Yield
______/ 2.5 pts Kinetic Characterization
______/ 2.5 pts Inhibition

Procedures _____/35 pts

C. Expt.3SDSPAGE:/10pts
/2.5ptsSDSPAGE
/2.5ptsProteinconcentration
/2.5ptsPreparation/running
/2.5ptsStaining/destaining
D. Expt.4YieldandSpecificActivityofLDH:/10pts
/2.5ptsAssayexplanationandreasonfor340nm
/2.5ptsSpecificactivityforchicken
/2.5ptsSpecificactivityforHistaggedLDH
/2.5ptsComparisonofchickenvs.HistaggedLDH
E. Expt.5KineticCharacterization:/10pts
/2.5ptsDeterminationofK
m
,V
max
,K
cat
w/lactate.
/5ptsDeterminationofprecisionofK
m
,V
max
,K
cat
/2.5ptsComparisonofLDHfromchickenandbacteria.(ifpossible)
F. Expt.6KineticCharacterization/Inhibition:/5pts
/2.5ptsDeterminationofK
m
app
,V
max
app
,K
I
w/oxalate.
/2.5ptsDeterminationofprecisionofK
m
app
,V
max
app
,K
I
w/oxalate
.

Results
Expt.3SDSPAGE:/15pts
____/5ptsSDSPAGEgelsketchesforchickenLDHandHistaggedLDH
____/5ptsSemilogplotforSDSPAGEgels
____/5ptsNative/Relativemolecularweightcalculations
Expt.4Yield:/20pts
____/ 5 pts Raw activity data for chicken LDH
Activity & specific activity calculations
Raw activity data plots (crude homogenate, desalted, purified)
Chicken specific activity & kcat
cat

/ 5 pts Raw activity data for His-tagged LDH bacteria
Activity & specific activity calculations
Raw activity data plots
Bacteria specific activity & kcat
cat

_____/ 5 pts Purification Table chicken LDH
_____/ 5 pts Purification Table chicken His-tagged LDH
Expt.57KineticCharacterization:/25pts
/5ptsRawdataplots
/5ptsTablesofS
0
andV
0
valuesw/andw/oinhibitor
/5ptsDirectlinearplots
/5ptsEstimatesofK
m
,V
max
,K
cat,
K
m
app
,V
max
app
,K
I
w/Precisions
/5ptsLineweaverBurkplots

Discussion ____/90 pts


M-125
Experiment #7: Crystallization of Proteins


Date Received:

J. Objective/Introduction
a. Brief description of crystallography and objectives.

II. Procedures
a. Step by step procedure.

III. Results/Data
a. Indicate your fraction 1 activity, concentration, and whether you used NADH or lactate in
the crystallization conditions.
b. Include a copy of your observations.
c. Include any sketches you made of crystals.

IV. Discussion
a. What is the best condition (pH, salt, precipitant) for the crystallization of your protein
sample?
b. What is function of salt and precipitant for protein crystallization. Which conditions
appear to be best based on total lab data?
c. What conclusions can you make based on the fraction 1 specific activities, concentration,
and the addition of either lactate or NADH?
d. Why would you add NADH into your protein sample for crystallization?
e. What are the reasons you may not crystallize the sample?

M-126
Experiment #8-9: Molecular Biology


Date Received:

I. ___/5 pts Objective/Introduction of each experiment and its purpose
A. Transformation of plasmid in bacteria.
B. Plasmid purification from E. coli
C. Restriction analysis of DNA
II.___/5 pts Procedures
Provide a detailed protocol for each experiment. Be sure to note all changes from the lab
manual
III. Results/Data
A. Transformation of plasmid DNA in to E. coli
1. ____/5 pts Bacterial growth curve
2. ____/5 pts Colonies
3. ____/5 pts Transformation efficiency (colonies/g of DNA)
B. Alkaline Lysis plasmid preparation from E. coli
1. ____/2.5 pts Absorbance of plasmid DNA preparation and fold dilution to obtain that
absorbance
2. ____/2.5 pts Concentration (mg/ml) and volume (ml or L) of plasmid containing
sample
3. ____/5 pts Total plasmid DNA yield (mg total plasmid/volume or gram starting
material)
C. Restriction analysis of DNA
1) ____/5 pts Indicate the amount of DNA you digested in each reaction.
2) ____/5 pts Fully labeled photo/photocopy/drawing of your DNA in agarose gel
picture
IV. Discussion
A. Transformation
1.____/5 pts Discuss your transformation efficiency and what it means
B. Alkaline Lysis Prep
1.____/5 pts Recovery
C. Restriction Analysis
1.____/10 pts Determine the size of digested and undigested Plasmid DNA as detailed
on page 10-10 of this experiment.
2.____10 pts Compare your own data with the expected sample results. What is
different and why? Is all the plasmid DNA digested?
Look up the Qiagen plasmid miniprep handbook on-line.
3.____/5 pts Label the sizes of the fragments accordingly on the picture of your DNA
gel.
Also, label the identity each band from the DNA gel on the circular map
4.____/10 pts Draw a circular map of the plasmid demonstrating the location and
estimated size of the plasmid and insert and the relative location of the

M-127
related restriction enzyme sites.
_____/90 Total Points

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BCH 102 Laboratory Manual

is the enthalpy of activation and S

in the rate-limiting step. This

in the transition state, through

in the transition state by

) also has lower

). The decrease in energy (G

of the catalytic reaction

, so that substrate binding is weak.

BCH 102 Laboratory Manual

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