Evonik - Agarose Gels For Proteins

The Biotechnology Education Company
111
EDVO-Kit #
Electrophoretic Properties of Native Proteins

Storage: See page 3 for specific storage instructions.
EXPERIMENT OBJECTIVES:
The objective of this experiment is to develop a general understanding of the structure and electrophoretic migration of native proteins.
All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
EVT 011285AM
EDVO-Kit # 111
Table of Contents
Page Experiment Components Experiment Requirements Background Information Electrophoretic Properties of Native Proteins Experiment Procedures Experiment Overview Preparations for Agarose Gel Electrophoresis Practice Gel Loading Conducting Agarose Gel Electrophoresis Staining the Gel Study Questions Instructor's Guidelines Notes to the Instructor Pre-Lab Preparations Quick Reference Tables Avoiding Common Pitfalls Idealized Schematic of Results Study Questions and Answers Material Safety Data Sheets 15 16 17 18 19 20 22 7 8 11 12 13 14 3 3
EVT 011285AM
The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
EDVO-Kit # 111
Experiment Components
A B C D E Bovine Serum Albumin (BSA) Ovalbumin Cytochrome C Lysozyme Horse Serum Proteins Practice Gel Loading Solution UltraSpec-Agarose Powder 50x Electrophoresis Buffer Protein InstaStain Sheets 1ml Pipet 100ml Graduated Cylinder (packaging for samples) Microtipped Transfer Pipets
Quick Reference:
There is enough sample for 6 gels if you are using an automatic micropipet for sample delivery. Use of transfer pipets will yield fewer gels.
Store Components A-E at 4C. This experiment contains ready-to-load protein samples and reagents sufficient for 6 gels (see Quick Reference).
Requirements
All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. UltraSpec-Agarose and Protein Plus are trademarks of EDVOTEK, Inc. Protein InstaStain, EDVOTEK, and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc. Horizontal Gel Electrophoresis Apparatus D.C. Power Supply Automatic Micropipets with Tips Waterbath Recommended Equipment: Visualization System (white light) Glass Staining Tray 250ml Flasks Pipet Pump Hot Gloves Marking Pens Distilled or Deionized Water Methanol Glacial Acetic Acid
EDVOTEK - The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com 24-hour FAX: (301) 340-0582 email: edvotek@aol.com
EVT 011285AM
EDVO-Kit # 111
Properties of Native Proteins

Proteins are a highly diversified class of biomolecules. Differences in their chemical properties, such as charge, shape, size and solubility, enable them to perform many biological functions. These functions include enzyme catalysis, metabolic regulation, binding and transport of small molecules, gene regulation, immunological defense and cell structure. A protein can have a net negative or positive charge, depending on its amino acid composition and pH conditions. At a certain pH, the molecule can be electrically neutral, i.e. negative and positive charges are equal. In this case, the protein is isoelectric. In the presence of an electrical field, a protein with a net negative or positive charge will migrate towards the electrode of opposite charge. The amino acid residues responsible for a proteins negative charge at physiological pH are glutamic acid and aspartic acid. A proteins positive charge at physiological pH is due to lysine, arginine and, to a lesser extent, histidine.
H+ | H-N-H O Glutamic Acid CH2- CH2- C OH H | + CH2- CH2- CH2- CH2- N - H | H C C-Terminal O HO O O C O O CH2- CH2- C OH H | + CH2- CH2- CH2- CH2- N - H | H C H+ | H-N-H O CH2- CH2- C OH H | CH2- CH2- CH2- CH2- N | H H+ | H-N-H O
Background Information
N-Terminal
Lysine
Acidic pH (<4)
Neutral pH (7)
Alkaline pH (>9)
Figure 1:
Effect of pH on Peptide Containing Glutamic and Lysine Residues At acidic pH, glutamic acid and aspartic acid residues have little charge, while lysine and arginine both have positive charges. As the pH of the protein solution is raised, glutamic and aspartic acid release a proton and become negatively charged. However, lysine and arginine residues become uncharged as the pH is raised to high values. (See Figure 1.) The direction and extent of a proteins migration in an electric field can be altered by using an acidic, neutral or alkaline buffer system during electrophoresis. The isoelectric point of a protein is defined as the pH at which the protein has no net charge. Consequently, a protein will not migrate in an electric field at its (pI) isoelectric point. An isoelectric protein still possesses areas of negative and positive charge, but overall
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright 1989,1992,1994,1995,1997, 1998, 1999, 2000, 2005 EDVOTEK, Inc., all rights reserved. EVT 011285AM The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
EDVO-Kit # 111

they cancel each other. Proteins that contain more aspartic and glutamic acid residues will have isoelectric points at acidic pH values. Conversely, proteins that contain more lysine and arginine residues will have isoelectric points at alkaline pH values. Some proteins contain other chemical groups in addition to their amino acid residues. For example, hemoglobin and cytochrome c contain heme, which consists of iron complexed to a system of organic rings called porphyrin. Non-amino acid structural groups covalently attached or very tightly bound to the polypeptide chain(s) of a protein are called prosthetic groups. Prosthetic groups enable proteins to perform biological functions and have a strong influence on the proteins chemical properties. The oxygen atoms transported by hemoglobin are actually bound at the heme irons. Proteins exhibit many different three-dimensional shapes and complex folding patterns which are determined by their amino acid sequence and post translational processing such as adding carbohydrate residues or prosthetic groups. The precise three-dimensional configuration of a protein is critical to its biological function. The general shapes for proteins are spherical, elliptical or rod-like. The molecular weight is a function of the number and type of amino acids in the polypeptide chain. Proteins can consist of a single polypeptide or several polypeptides specifically associated with each other. These polypeptides can be identical, similar or completely different from one another. The number and nature of polypeptides in a protein has large effects on its mass, size and shape. Proteins that are in their normal, biologically active forms are called native. Certain detergents, extremes of pH, organic solvents and heat can cause a native protein to lose its specific three-dimensional folding pattern and, consequently, its biological activity. Proteins that have gone through this process are called denatured. Denatured proteins can have radically different behavior from their native forms during electrophoresis.
ELECTROPHORESIS OF PROTEINS
The properties of proteins affect the way they migrate during gel electrophoresis. Gels used in electrophoresis (e.g. agarose, polyacrylamide) consist of microscopic pores of a defined size range that act as a molecular sieve. Only molecules with net charge will migrate through the gel when it is in an electric field. Small molecules pass through the pores more easily than large ones. Molecules having more charge than others of the same shape and size will migrate faster. Molecules of the same mass and charge can have different shapes. In this case, those with a more compact shape, like a sphere, will migrate through the gel more rapidly than those with an elongated shape, like a rod. In summary, the amount and sign of charge, the size and shape of a native protein, all affect its electrophoretic migration rates.
EDVO-Kit # 111

Electrophoresis of native proteins is useful in clinical and immunological analysis of complex biological samples, such as serum. Serum consists of many different types of proteins. Gel electrophoresis of native serum proteins at alkaline pH results in several zones. Albumin is by far the most abundant serum protein and has one of the fastest electrophoretic migration rates. Albumin binds and transports many small molecules, including fatty acids and bilirubin. It is also involved with osmotic regulation. The serum proteins with the slowest migration rates are the gamma globulins (antibodies). In between these two zones several other types of proteins can be observed. These include transferrin (iron transport), ceruloplasmin (copper transport), macroglobulin (protease inhibitor) and haptoglobin (involved with the binding and conservation of hemoglobin). Electrophoretic patterns of human serum proteins can aid in the diagnosis of certain diseases. For instance, cirrhosis of the liver causes a decrease in albumin, while multiple myeloma ( a cancer of the immune system) and chronic rheumatoid arthritis causes abnormal increases in the gamma globulins. The purified proteins used in this experiment all consist of single polypeptide chains, but differ in their charge and mass. Bovine serum albumin, with a molecular weight of 68,000 and an isoelectric point (pI) of 4.7, is similar in structure and function to the serum albumin previously discussed. Ovalbumin is an abundant protein found in eggs. Ovalbumin, with a molecular weight of 43,000 and pI of 4.6, contains covalently attached carbohydrate and is therefore a glycoprotein. The polysaccharide chain contains eight (8) sugar residues. The majority of serum proteins are also glycoproteins (except serum albumin). Cytochrome C, a ubiquitous protein with a molecular weight of 12,000 and pI of 10.7, is involved in electron transport reactions that are mediated by the heme prosthetic group. Cytochrome C and several other proteins form the electrontransport chain which enables the cell to obtain chemical energy (ATP) from the oxidation of glucose. In bacteria, cytochrome c is located on the inner surface of the cellular membrane. Lysozyme, a small protein with a molecular weight of 14,000 and pI of11.2, is utilized in bacterial cell lysis. All these proteins are dissolved in a buffer containing glycerol and the negatively charged bromophenol blue tracking dye. This tracking dye generally migrates faster than the proteins. During the electrophoresis of the serum proteins, the bromophenol blue may separate into two bands. This separation occurs because some of the serum proteins bind a fraction of the dye. The bound dye has a slower migration rate than the free dye. After electrophoresis, the proteins will be visualized by staining with Protein InstaStain After the gel is destained, proteins will appear as dark blue zones against a light blue background.
EDVO-Kit # 111
Experiment Overview
EXPERIMENT OBJECTIVE:
The objective of this experiment module is to develop a general understanding of the structure and electrophoretic migration of native proteins.
Experiment Procedures
LABORATORY SAFETY
Wear gloves and safety goggles
1. Gloves and goggles should be worn routinely as good laboratory practice. Exercise extreme caution when working with equipment which is used in conjunction with the heating and/or melting of reagents. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS OR BULBS.
2.
3.
EDVO-Kit # 111
Preparations for Agarose Gel Electrophoresis
PREPARING THE GEL BED
1. 2.
Make sure the gel bed is clean and dry. Close off the open ends of the bed by using rubber dams or tape. A. Using Rubber dams: Place a rubber dam on each end of the bed. Make sure the rubber dam sits firmly in contact with the sides and bottom of the bed. Taping with labeling or masking tape: With 3/4 inch wide tape, extend the tape over the sides and bottom edge of the bed. Fold extended edges of the tape back onto the sides and bottom. Press contact points firmly to form a good seal.
B.
3.
Place the well forming template (comb) across the bed in the middle set of notches. The comb should sit firmly and evenly across the bed.
CASTING THE AGAROSE GEL

This experiment requires a 0.8% gel. 3. Use a 250ml flask to prepare the diluted gel buffer. 4. 5. With a 1ml pipet, measure the buffer concentrate and add the distilled water as indicated in Table A.
Add the required amount of agarose powder. Swirl to disperse clumps. With a marking pen, indicate the level of the solution volume on the outside of the flask.
Table A
Size of EDVOTEK Casting Tray (cm)
Individual 0.8% UltraSpec-Agarose Gel

Amt of Agarose (g) + Distilled Total Concentrated Buffer (50x) + Water = Volume (ml) (ml) (ml)
7 x 15
0.48
1.2
58.8
60
EDVO-Kit # 111

6. Heat the mixture to dissolve the agarose powder. The final solution should be clear (like water) without any undissolved particles.
A. Microwave method: Cover flask with plastic wrap to minimize evaporation. Heat the mixture on high for 1 minute. Swirl the mixture and heat on high in bursts of 25 seconds until all the agarose is completely dissolved. B. Hot plate or burner method: Cover the flask with foil to prevent excess evaporation. Heat the mixture to boiling over a burner with occasional swirling. Boil until all the agarose is completely dissolved.
7.
Cool the agarose solution to 60C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step 5.
60C
After the gel is cooled to 60C:

If using rubber dams, go to step 9. If using tape, continue with step 8. 8. Seal the interface of the gel bed and tape to prevent the agarose solution from leaking. Use a transfer pipet to deposit a small amount of cooled agarose to both inside ends of the bed. Wait approximately 1 minute for the agarose to solidify. Pour the cooled agarose solution into the bed. Make sure the bed is on a level surface.
Caution!
DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED. Hot agarose solution may irreversibly warp the bed. 9.
10. Allow the gel to completely solidify. It will become firm and cool to the touch after approximately 20 minutes.
10
EDVO-Kit # 111
PREPARING THE SOLIDIFIED GEL FOR ELECTROPHORESIS

11. Carefully remove the rubber dams or tape. 12. Remove the comb by slowly pulling it straight up. Do this carefully and evenly to prevent tearing the sample wells. 13. Inspect the wells by viewing the gel from the edge nearest the wells. If some of the wells are ripped through their bottoms or sides, do not use them when loading samples. 14. Place the gel in the electrophoresis chamber, properly oriented, centered and level on the platform. 15. Fill the chamber of the electrophoresis apparatus with the required volume of diluted buffer as outlined in Table B. 16. Load samples in wells; conduct electrophoresis according to experiment instructions. See Table C for time and voltage guidelines.
Useful Hint!
Table B
EDVOTEK Model #
Electrophoresis Buffer
Distilled Concentrated Buffer (50x) + Water (ml) (ml) Total = Volume (ml)
Step 11: Be careful not to damage or tear the gel when removing rubber dams. A thin plastic knife or spatula can be inserted between the gel and the dams to break possible surface tension.
M6+ M12 M36 (blue) M36 (clear)
6 8 10 20
294 392 490 980
300 400 500 1000
EDVO-Kit # 111
11
Practice Gel Loading
Quick Reference:
If you are using an automatic micropipet, deliver 20 microliters to the sample well. If using transfer pipets, load the sample well until it is full.
EDVOTEK experiments which involve electrophoresis contain practice gel loading solution. If your students are unfamiliar with loading samples in agarose gels, it is suggested that they practice sample delivery techniques before performing the electrophoresis part of an experiment. Using the EDVOTEK system, sample delivery can be performed by using either an automatic micropipet, or disposable microtipped transfer pipets. Casting of a separate practice gel is highly recommended. One suggested activity for practice gel loading is outlined below: 1. Cast a gel with the maximum number of wells and place it under the buffer in an electrophoresis apparatus chamber. (Use microbiology-grade agar and water to make practice gels - save the agarose for the experiment.) Let students practice delivering the practice gel loading solution to the sample wells. If students need more practice, remove the practice gel loading solution by squirting buffer into the wells with a transfer pipet. When students are finished practicing, replace the practice gel with a fresh gel and continue with the experiment. The practice gel loading solution will become diluted in the buffer and will not interfere with the experiment.
Remember!
Use microbiology-grade agar and water to make practice gels - save the agarose for the experiment. 2.
3.
4.
12
EDVO-Kit # 111
Conducting Agarose Gel Electrophoresis
Reminder:
During electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode. Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.
Black Sample wells
This experiment requires a 0.8% UltraSpec-Agarose gel. The gel should be cast with the comb placed in the middle set of notches of the gel bed. Make sure the electrophoresis apparatus leads reach the power source before loading samples. The apparatus should not be moved after the samples are loaded because movement of the unit will cause samples to spill out of the wells.
LOADING PROTEIN SAMPLES

1. Consecutively load 40l of each sample in tubes A - E into wells in the middle of the gel.
+ Red
RUNNING THE GEL

tro Elec esis phor 2 M1
2.
After the samples are loaded, carefully snap the cover down onto the electrode terminals. Make sure that the negative and positive indicators on the cover and apparatus chamber are properly oriented.
Table C:
Volts
Time and Voltage

Optimal
Recommended Time
Minimum
3.
125 70 50
30 min 40 min 60 min
45 min 1.5 hrs 2.0 hrs

4.
Insert the plug of the black wire into the black input of the power source (negative input). Insert the plug of the red wire into the red input of the power source (positive input). Set the power source at the required voltage and run the electrophoresis for the length of time as determined by your instructor. When current is flowing properly, you should see bubbles forming on the electrodes. After the electrophoresis is completed, turn off the power, unplug the power source, disconnect the leads and remove the cover.
Useful Hint!
After electrophoresis, remove a small slice of the gel from the upper right hand corner to easily identify right and left orientation of the gel after staining. 5.
EDVO-Kit # 111
13
Staining the Gel

ONE-STEP STAINING & DESTAINING WITH PROTEIN INSTASTAIN
Protein agarose gels can be stained with Protein InstaStain cards in one easy step an excellent alternative if time does not permit staining during a regular class session. 1. After electrophoresis, submerge the gel and plate in a small tray with 100ml of fixative solution. (Use enough solution to cover the gel.) Gently float a sheet of Protein InstaStain with the stain side (blue) in the liquid. Cover the gel to prevent evaporation. Gently agitate on a rocking platform for 1-3 hours or overnight. After staining, protein bands will appear as dark blue bands against a light background and will be ready for photography. NO DESTAINING IS REQUIRED. If the gel is too dark, destain in several changes of fresh destain solution until the appearance and contrast of the protein bands against the background improves.
2.
3. 4.
5.
Fixative and Destaining Solution for each gel (100ml) 50ml 10ml 40ml Methanol Glacial Acetic Acid Distilled Water
14
EDVO-Kit # 111
Study Questions
1. Under the conditions of the electrophoresis (pH 7.8), which of the proteins (BSA, ovalbumin, Cytochrome C, Lysozyme) are net positive? Which are net negative? Based on your observations, would you expect ovalbumin to have more or less lysine and arginine residues than lysozyme? Why? A sample of native protein is submitted to native gel electrophoresis. After the electrophoresis was run for several hours, it was found that the protein did not enter the gel from the sample well (did not migrate). What is the most likely explanation for this observation? What experimental condition could you change that would allow the protein to migrate? Consider the following information about the proteins used in this experiment: Approximate Isoelectric Protein Molecular Weight Point (pH) Cytochrome C 12,000 10.7 Lysozyme 14,000 11.2 Ovalbumin 43,000 4.6 BSA 68,000 4.7 All these proteins are spherical in shape, therefore, increasing molecular weight corresponds to increasing size. The results of the electrophoresis at pH 7.8 should show that ovalbumin migrates a greater distance towards the positive electrode than BSA. The results should also show that Lysozyme migrates a greater distance towards the negative electrode than Cytochrome C. Explain the results using the information provided above.
2.
3.
4.
EDVO-Kit # 111
15
Notes to the Instructor

If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at 1-800EDVOTEK (1-800-338-6835).
Instructor's Guide
Online Ordering now available

www.edvotek.com
APPROXIMATE TIME REQUIREMENTS FOR PRE-LAB AND EXPERIMENTAL PROCEDURES

1. Agarose gel preparation: Your schedule will determine when to prepare the agarose gel(s). Whether you choose to prepare the gel(s), or have the students do it, allow approximately 30 to 40 minutes for this procedure. Generally, 20 minutes of this time is required for gel solidification. The approximate time for electrophoresis will vary from 45 minutes to 2 hours. A variety of factors, such as class size, length of laboratory sessions, and availability of equipment, will influence the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances.
Visit our web site for information about EDVOTEK's complete line of experiments for biotechnology and biology education.
2.
Technical Service Department Mon - Fri 9:00 am to 6:00 pm ET

FAX: (301) 340-0582 web: www.edvotek.com email: edvotek@aol.com
ED
-T VO
E C H S E RV I C E
1-800-EDVOTEK
(1-800-338-6835)
SPECIFIC REQUIREMENTS FOR THIS EXPERIMENT

Gel Concentration
This experiment requires a 0.8% UltraSpecAgarose gel.
Mo
pm - 6 Please have the following information: n - Fri 9 am The experiment number and title Kit Lot number on box or tube The literature version number (in lower right corner) Approximate purchase date
ET
Number of Wells Required

This experiment requires a gel with 5 sample wells.
Placement of Comb
During gel casting, the comb should be placed in the notches in the middle of the gel bed.
16
EDVO-Kit # 111
PreLab Preparations
PREPARING THE ELECTROPHORESIS (CHAMBER) BUFFER
Instructor's Guide
Useful Hint!
Prepare the appropriate volume of diluted electrophoresis chamber buffer by mixing the concentrated 50x electrophoresis buffer and distilled or deionized water according to Table B .
The same 50x concentrated buffer is used for preparing the agarose gel buffer and the chamber buffer.
ELECTROPHORESIS TIME AND VOLTAGE

Your schedule will dictate the length of time samples will be separated by electrophoresis. General guidelines are presented in Table C.
PREPARING STAINING AND DESTAINING SOLUTIONS

Solution for staining with Protein InstaStain
Prepare a stock solution of Methanol and Glacial Acetic Acid by combining 180ml Methanol, 140ml Distilled water, and 40 ml Glacial Acetic Acid. No destaining is required.
EDVO-Kit # 111
17
Quick Reference Tables
Instructor's Guide
Table A
Size of EDVOTEK Casting Tray (cm)
Individual 0.8% UltraSpec-Agarose Gel

Amt of Agarose (g) + Distilled Total Concentrated Buffer (50x) + Water = Volume (ml) (ml) (ml)
7 x 15
0.48
1.2
58.8
60
Table B
EDVOTEK Model #
Electrophoresis Buffer
Distilled Concentrated Buffer (50x) + Water (ml) (ml) Total = Volume (ml)
M6+ M12 M36 (blue) M36 (clear)
6 8 10 20
294 392 490 980
300 400 500 1000
Table C
Volts
Time and Voltage Electrophoresis of DNA

Recommended Time Maximum Minimum
125 30 min 70 50 40 min 60 min
45 min 1.5 hrs 2.0 hrs
18
EDVO-Kit # 111
Avoiding Common Pitfalls

To ensure that protein bands are well resolved, make sure the gel formulation is correct (see Table A on page 17) and that electrophoresis is conducted for the optimum recommended amount of time. Correctly dilute the concentrated buffer for preparation of the gel and electrophoresis buffer. Remember that without buffer in the gel, there will be no protein mobility. Use only distilled water to prepare buffers. Use distilled or deionized water. For optimal results, use fresh electrophoresis buffer prepared according to instructions. Before performing the actual experiment, practice sample delivery techniques to avoid diluting the sample with buffer during gel loading. To avoid loss of protein bands into the buffer, make sure the gel is properly oriented so the samples are not moving in the wrong direction and off the gel. If protein bands appear faint after staining and destaining, repeat the staining procedure but stain for a longer period of time.
Instructor's Guide
EDVO-Kit # 111
19
Idealized Schematic of Results
(-) 1 2 3 4 5 6
The figure to the left is an idealized schematic showing relative positions of protein polypeptides. The idealized schematic (left) shows the relative positions of the bands, but are not depicted to scale. Actual results are shown below. Lane 1 2 3 4 5 Tube A B C D E Bovine Serum Albumin (BSA) Ovalbumin Cytochrome C Lysozyme Horse Serum Proteins
Instructor's Guide
(-) 1 2 3 4 5 6
(+)
(+)
20
EDVO-Kit # 111
Study Questions and Answers

1. Under the conditions of the electrophoresis (pH 7.8), which of the proteins (BSA, ovalbumin, Cytochrome C, Lysozyme) are net positive? Which are net negative? At pH 7.8 cytochrome c (pI 10.7) and lysozyme (pI 11.2) have a net positive charge and therefore travel toward the negative electrode. BSA (pI 4.7) and ovalbumin (pI 4.6) are negative and therefore travel to the positive electrode. 2. Based on your observations, would you expect ovalbumin to have more or less lysine and arginine residues than lysozyme? Why? Ovalbumin has less Lysine and Arginine residues and therefore is less positive (more negative) and thus moves toward the positive electrode (anode). 3. A sample of native protein is submitted to native gel electrophoresis. After the electrophoresis was run for several hours, it was found that the protein never entered the gel from the sample well, i.e. it did not migrate. What is the most likely explanation for this observation? What experimental condition could you change that would allow the protein to migrate? The buffer used for separation is pH 7.8. Assume that the proteins are spherical in shape and therefore conformation will not be a factor, and the molecular weight will correspond directly to the size. Ovalbumin has a molecular weight of 43,000 and a pI of 4.6. Therefore at pH 7.8, it will be negative. BSA has larger molecular weight 68,000 with approximately the same pI of 4.7. Therefore both ovalbumin and BSA have a net negative charge, but since BSA is larger it will move slower. 4. Consider the following information on the proteins used in this experiment: Approximate Isoelectric Protein Molecular Weight Point (pH) Cytochrome C 12,000 10.7 Lysozyme 14,000 11.2 Ovalbumin 43,000 4.6 BSA 68,000 4.7 All these proteins are spherical in shape, therefore, increasing molecular weight corresponds to increasing size. The results of the electrophoresis at pH 7.8 should show that Ovalbumin migrates a greater distance towards the positive electrode than BSA. The results should also show that Lysozyme migrates a greater distance towards the negative electrode than Cytochrome C. Explain the results using the information provided above. (Answer on next page.)
Instructor's Guide
EDVO-Kit # 111
21
Study Questions and Answers

The following are possible explanations:
Instructor's Guide
A. The agarose gel pores are not large enough. Therefore, protein samples cannot penetrate the gel. B. The protein has a net charge of zero at pH 7.8, the pH of the electrophoresis buffer.
Possible remedies: Decrease the gel concentration to make larger pores. Change the pH of the electrophoresis buffer.
Section V - Reactivity Data

Material Safety Data Sheet
May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements.
Stability
Incompatibility
Unstable Stable
Conditions to Avoid
None
No data available
IDENTITY (As Used on Label and List)
Agarose
Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.
Hazardous Decomposition or Byproducts
Section I
Manufacturer's Name Emergency Telephone Number
Telephone Number for information
Hazardous Polymerization
May Occur Will Not Occur
Conditions to Avoid
None
Skin? Ingestion?
EDVOTEK, Inc.
Address (Number, Street, City, State, Zip Code)
(301) 251-5990 (301) 251-5990
Section VI - Health Hazard Data

Route(s) of Entry: Inhalation?
Yes
Yes
Yes
14676 Rothgeb Drive Rockville, MD 20850
Date Prepared
Health Hazards (Acute and Chronic)
09-15-2002
Carcinogenicity:
Inhalation: No data available

NTP?
Ingestion: Large amounts may cause diarrhea

IARC Monographs? OSHA Regulation?
Signature of Preparer (optional)
Signs and Symptoms of Exposure
No data available No data available
Section II - Hazardous Ingredients/Identify Information

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Other Limits Recommended
Medical Conditions Generally Aggravated by Exposure

% (Optional)
This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. CAS #9012-36-6
Emergency First Aid Procedures
Treat symptomatically and supportively
Section III - Physical/Chemical Characteristics

Boiling Point
Section VII - Precautions for Safe Handling and Use

Steps to be Taken in case Material is Released for Spilled
For 1% solution
194 F No data No data
Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1)
No data No data No data

Waste Disposal Method
Sweep up and place in suitable container for disposal
Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Solubility in Water Appearance and Odor
Normal solid waste disposal

Precautions to be Taken in Handling and Storing
Insoluble - cold White powder, no odor N.D. = No data

LEL
None
Other Precautions
None
Flammable Limits UEL
Section IV - Physical/Chemical Characteristics

Flash Point (Method Used) Extinguishing Media
Section VIII - Control Measures

N.D.
Respiratory Protection (Specify Type) Ventilation Protective Gloves
No data
N.D.
Chemical cartridge respirator with full facepiece.

Special
Water spray, dry chemical, carbon dioxide, halon or standard foam
Local Exhaust
Special Fire Fighting Procedures
Mechanical (General)Gen. dilution ventilationOther
Possible fire hazard when exposed to heat or flame

Unusual Fire and Explosion Hazards
Yes
Eye Protection
Splash proof goggles
Other Protective Clothing or Equipment
None
Work/Hygienic Practices
Impervious clothing to prevent skin contact None
Section V - Reactivity Data Material Safety Data Sheet
Stability
Incompatibility
Unstable Stable
Conditions to Avoid
None
Strong oxidizing agents Carbon monoxide, Carbon dioxide

Conditions to Avoid
50x Electrophoresis Buffer
Section I
Manufacturer's Name Emergency Telephone Number
May Occur Will Not Occur Inhalation?
None
Skin? Ingestion? Yes
EDVOTEK, Inc.
(301) 251-5990 (301) 251-5990

Route(s) of Entry:
Yes
Yes
Health Hazards (Acute and Chronic) Carcinogenicity: None identified Signs and Symptoms of Exposure
Date Prepared Signature of Preparer (optional)
None
NTP? IARC Monographs? OSHA Regulation?
09-15-02
Irritation to upper respiratory tract, skin, eyes None

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Other Limits Recommended % (Optional)
Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures
This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard.
Ingestion: If conscious, give large amounts of water
Eyes: Flush with water
Inhalation: Move to fresh air Skin: Wash with soap and water

Boiling Point Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Solubility in Water Appearance and Odor

Steps to be Taken in case Material is Released for Spilled Wear suitable protective clothing. Mop up spill
and rinse with water, or collect in absorptive material and dispose of the absorptive material.
Dispose in accordance with all applicable federal, state, and local enviromental regulations.
Appreciable, (greater than 10%) Clear, liquid, slight vinegar odor

Other Precautions
Avoid eye and skin contact.
None

Flash Point (Method Used)
N.D. = No data
LEL UEL

N.D.
Respiratory Protection (Specify Type) Ventilation Protective Gloves Local Exhaust
Flammable Limits
No data
Extinguishing Media Special Fire Fighting Procedures
N.D.
Use extinguishing media appropriate for surrounding fire. Wear protective equipment and SCBA with full facepiece operated in positive pressure mode.
Yes Yes
Special
None None
Mechanical (General)
Other
Yes None None
Safety goggles Eye Protection
Other Protective Clothing or Equipment Unusual Fire and Explosion Hazards
None identified
Work/Hygienic Practices
Stability
Incompatibility
Unstable Stable
Conditions to Avoid
None
None Sulfur oxides, and bromides

Conditions to Avoid
Practice Gel Loading Solution Section I

Manufacturer's Name
None
Skin? Ingestion? Yes
Emergency Telephone Number

EDVOTEK, Inc.
(301) 251-5990 (301) 251-5990

Route(s) of Entry:
Yes
Yes
Date Prepared
Health Hazards (Acute and Chronic) Carcinogenicity:
09-19-2002
No data available
Signs and Symptoms of Exposure
Acute eye contact: May cause irritation. No data available for other routes.
May cause skin or eye irritation None reported

Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures
This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard.
Treat symptomatically and supportively. Rinse contacted area with copious amounts of water.


Wear eye and skin protection and mop spill area. Rinse with water.
Observe all federal, state, and local regulations.

Soluble Blue liquid, no odor
Avoid eye and skin contact.

Other Precautions
None
LEL UEL

Flash Point (Method Used) Extinguishing Media

Respiratory Protection (Specify Type) Ventilation Protective Gloves Local Exhaust Mechanical (General)
No data
Flammable Limits
No data
No data
Dry chemical, carbon dioxide, water spray or foam
Yes Yes
Special
None None Splash proof goggles
Other
Use agents suitable for type of surrounding fire. Keep upwind, avoid breathing hazardous sulfur oxides and bromides. Wear SCBA. Unknown
Yes None required
Eye Protection
Unusual Fire and Explosion Hazards
Other Protective Clothing or Equipment Work/Hygienic Practices
Avoid eye and skin contact
Stability
Incompatibility
Unstable Stable
Conditions to Avoid
None
Strong oxidizing agents Carbon monoxide, Carbon dioxide, Sulfur oxides

Conditions to Avoid
Protein Plus Stain Section I

Manufacturer's Name
None
Skin? Ingestion?

EDVOTEK, Inc.
(301) 251-5990 (301) 251-5990

Route(s) of Entry: Health Hazards (Acute and Chronic) Carcinogenicity: Signs and Symptoms of Exposure
Date Prepared
Yes Yes Yes Irritating to eyes, skin, mucous membranes and upper respiratory tract. Chronic exposure may cause lung damage or pulmonary sensitization
09-19-2002
No data
No data
No data

Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache
No data
Methanol (Methyl Alcohol) 200ppm 200ppm No data 90%-100% CH3OH
Flush skin/eyes w/ large amounts of water. If inhaled, remove to fresh air. Ingestion: give large amounts of water or milk. Do not induce vomiting.


65C 96mmHg 1.11 Complete (100%)
.79 N/A 4.6
Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up w/ absorptive material and burn in chemical incinerato equipped w/ an afterburner and scrubber.
Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation.

Other Precautions
Blue liquid/alcoholic, pungent odor
Strong sensitizer
LEL UEL

Flash Point (Method Used) Flammable Limits

(closed cup) 12C

Extinguishing Media
6.0%
36%
NIOSH/MSHA approved respirator No No

Special
Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective)
Local Exhaust Mechanical (General)
Chem fume hood None Splash-proof goggles
Other Eye Protection
Wear SCBA with full facepiece operated in positive pressure mode. Move containers from firearea Unusual Fire and Explosion Hazards Vapors may flow along surfaces to distant ignition sources. Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire.
Rubber Rubber boots
Avoid prolonged or repeated exposure
Stability
Incompatibility
Unstable Stable
Conditions to Avoid
None
Strong oxidizing agents Carbon monoxide, Carbon dioxide, Sulfur oxides

Conditions to Avoid
Protein InstaStain Section I

Manufacturer's Name
None
Skin? Ingestion?

EDVOTEK, Inc.
(301) 251-5990 (301) 251-5990

Route(s) of Entry: Health Hazards (Acute and Chronic) Carcinogenicity: Signs and Symptoms of Exposure
Date Prepared
09-19-2002
Yes Yes Yes Irritating to eyes, skin, mucous membranes and upper respiratory tract. Chronic exposure may cause lung damage or pulmonary sensitization
No data
No data
No data

Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache
No data
Methanol (Methyl Alcohol) 200ppm 200ppm No data 90%-100% CH3OH
Flush skin/eyes w/ large amounts of water. If inhaled, remove to fresh air. Ingestion: give large amounts of water or milk. Do not induce vomiting.


65C 96mmHg 1.11 Complete (100%)
.79 N/A 4.6
Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up w/ absorptive material and burn in chemical incinerato equipped w/ an afterburner and scrubber.
Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation. chemical bound to paper, no odor

Other Precautions
Strong sensitizer
LEL UEL

Flash Point (Method Used) Flammable Limits

(closed cup) 12C

Extinguishing Media
6.0%
36%
NIOSH/MSHA approved respirator No No

Special
Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective)
Local Exhaust Mechanical (General)
Chem fume hood None Splash-proof goggles
Other Eye Protection
Wear SCBA with full facepiece operated in positive pressure mode. Move containers from firearea Unusual Fire and Explosion Hazards Vapors may flow along surfaces to distant ignition sources. Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire.
Rubber Rubber boots
Avoid prolonged or repeated exposure

Evonik - Agarose Gels For Proteins

Caricato da

Informazioni sul documento

Descrizione originale:

Titolo originale

Copyright

Formati disponibili

Condividi questo documento

Condividi o incorpora il documento

Opzioni di condivisione

Hai trovato utile questo documento?

Questo contenuto è inappropriato?

Copyright:

Formati disponibili

Evonik - Agarose Gels For Proteins

Caricato da

Copyright:

Formati disponibili

The Biotechnology Education Company

Electrophoretic Properties of Native Proteins

Electrophoretic Properties of Native Proteins

The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com

Electrophoretic Properties of Native Proteins

Electrophoretic Properties of Native Proteins

Properties of Native Proteins

Electrophoretic Properties of Native Proteins

Properties of Native Proteins

Electrophoretic Properties of Native Proteins

Properties of Native Proteins

Electrophoretic Properties of Native Proteins

Electrophoretic Properties of Native Proteins

Preparations for Agarose Gel Electrophoresis

PREPARING THE GEL BED

Wear gloves and safety goggles

CASTING THE AGAROSE GEL

Individual 0.8% UltraSpec-Agarose Gel

Electrophoretic Properties of Native Proteins

Preparations for Agarose Gel Electrophoresis

After the gel is cooled to 60C:

Electrophoretic Properties of Native Proteins

Preparations for Agarose Gel Electrophoresis

PREPARING THE SOLIDIFIED GEL FOR ELECTROPHORESIS

M6+ M12 M36 (blue) M36 (clear)

294 392 490 980

300 400 500 1000

Electrophoretic Properties of Native Proteins

Practice Gel Loading

Electrophoretic Properties of Native Proteins

Conducting Agarose Gel Electrophoresis

LOADING PROTEIN SAMPLES

RUNNING THE GEL

Time and Voltage

30 min 40 min 60 min

45 min 1.5 hrs 2.0 hrs

Electrophoretic Properties of Native Proteins

Staining the Gel

Electrophoretic Properties of Native Proteins

Electrophoretic Properties of Native Proteins

Notes to the Instructor

Online Ordering now available

APPROXIMATE TIME REQUIREMENTS FOR PRE-LAB AND EXPERIMENTAL PROCEDURES

Technical Service Department Mon - Fri 9:00 am to 6:00 pm ET

SPECIFIC REQUIREMENTS FOR THIS EXPERIMENT

Number of Wells Required

Electrophoretic Properties of Native Proteins

ELECTROPHORESIS TIME AND VOLTAGE

PREPARING STAINING AND DESTAINING SOLUTIONS

Wear gloves and safety goggles

Electrophoretic Properties of Native Proteins

Quick Reference Tables

Individual 0.8% UltraSpec-Agarose Gel

M6+ M12 M36 (blue) M36 (clear)

294 392 490 980

300 400 500 1000

Time and Voltage Electrophoresis of DNA

125 30 min 70 50 40 min 60 min

45 min 1.5 hrs 2.0 hrs

Electrophoretic Properties of Native Proteins

Avoiding Common Pitfalls

Electrophoretic Properties of Native Proteins