the process of preparing To separate

matrix fraction III (Invertase) through ion exchange

DEAE-Cellulose

OBJECTIVE

chnique of taking the absorbance using quartz cuvete.the elution buffer

To prepare
 INTRODUCTIO
N
 ION EXCHANGE
CHROMATOGRAPHY
• Process of separation of ions and
polar molecules based on their
charge.
• Eg : large proteins, small nucleotides,
and amino acids.
• Usually used in protein purification,
water analysis and quality control.
•
 ION EXCHANGE
• Exchange of ions between two
electrolytes.
• Mostly used to denote the purification,
and separation process.
• Reversible process.
• Ion exchanger : cations / anions /
amphoteric
• Anion exchangers exchange negatively
charged ions.
• Cations exchangers exchange the
positively charged ions.
 ION EXCHANGE
• Amphoteric exchangers, are able to
exchange both cations and anions
simultaneously.However, the
simultaneous exchange of cations and
anions can be more efficiently
performed in mixed beds that contain
a mixture of anion and cation
exchange resins.
• Ions exchanger are able to be
regenerated / loaded with desirable
ions by washing with an excess of
PRINCIPLES OF ION EXCHANGE
CHROMATOGRAPHY
• Relies on charge-charge interactions
between the proteins in the sample
and the charges immobilized on the
resin that has been chosen.
• Ion exchange chromatography can be
divided into
 - Cation exchange chromatography ;
positively charged ions bind to a
negatively charged resin
 - Anion exchange chromatography ;
the binding ions are negative, and the
PRINCIPLES OF ION EXCHANGE
CHROMATOGRAPHY
• As the solutes bounded, column is
washed to equilibrate the solutes in
the starting buffer (should be of low
ionic strength).
• Then, the bound molecules were eluted
off using gradient of second buffer
that increases the ionic strength of
the eluent solution.
• Note that, the pH of eluent buffe might
be modified to give the protein /
matrix charge at which, they would
not interact and the interest molecule
PRINCIPLES OF ION EXCHANGE
CHROMATOGRAPHY
• If you know the pH you want to run at and
need to decide what type of ion exchange
to use paste your protein sequence into
the titration curve generator. If it is
negatively charged at the pH you wish,
use an anion exchanger; if it is positive,
use a cation exchanger.
• This means that protein will be binding
under the conditions you choose.
• However, it might be more advantageous to
actually select conditions at which the
protein will flow through while the
contaminants will bind. “flow through
mode”
 MATERIALS
ØDiethylaminoethyl-cellulose(DE 23)
Ø
Ø0.50M Hydrochloric acid(HCl)
Ø
Ø0.50m Sodium hydroxide(NaOH)
Ø
Ø0.05M Tris buffer
Ø
ØSodium Chloride(NaCl)
 PREPARATION OF TRIS
BUFFER
• n =MV
 = 0.05 M × 1L
 = 0.05 mol
• Mass= 0.05 mol × 121.14
 = 6.057 g


Top up distilled water up to 1 L


 PREPARATION OF HCl
• No of moles = 0.50 M × 1L
 = 0.50 mol
• MASS = 0.50 mol ÷ 36.46
 = 18.23 g
• PURITY = 18.23 g ÷ 37/100
 = 49.27 g
 = 4.927 × 10 -2 kg ÷ 1.19 kg
 1L
 = 0.0414 L


=41.4 ml

Top up to 1 L


 PREPARATION OF NaCl
vn= MV
 = (200 × 10 ̄³) × 0.50 L
 = 0.1 mol
vMass = 0.1 ÷ 58.5
 = 5.85 g


Top up to 500 ml

 Preparation of B(i to v)
solution
 M1V1 = M2V2


i 20mM × 50 ml = 200 × V2
 V2 = 5 ml


ii . 40mM × 50 ml = 200 × V2

 V2= 10 ml


iii. 60mM × 50 ml = 200 × V2

 V2= 15 ml

iv . 80mM × 50 ml = 200 × V2


 V2 = 20 ml


v . 100mM × 50 ml = 200 × V2


 V2= 25 ml


• Top up with Tris buffer up to 50ml

A) Preparation of DEAE-
cellulose matrix
10g dry DE23-weight

200ml (0.50M)HCI added

Stir for 1 hour

Let matrix precipitated



 water removed


 Steps was repeated



 500ML distilled water added-stir



 Water removed


 Steps were repeated-water show ph~4.0



 200ML(0.50M) NAOH added-stir-1 hour




 Let matrix precipitated


 Water was removed



 Wash DE23 matrix until ph water 8.0



 DE23 matrix-keep in distilled

water



B)Preparation of elution
buffer
Prepare:
a. Initial buffer 0.05M Tris pH 7.3 (1L)
b. Initial buffer contain 200mM NaCl
(500mL)
c. Dilute (b) with initial buffer to obtain
buffer solution :
iv.20mM NaCl in initial buffer (50mL)
v. 40mM NaCl in initial buffer (50mL)
vi.60mM NaCl in initial buffer (50mL)
vii.
viii.80mM NaCl in initial buffer (50mL)
ix.100mM NaCl in initial buffer (50mL)
 C) Separation of F3 by ion exchange
chromatography
•
• F3 is dissolved with 5 mL initial buffer
 1 mL solution is taken to estimate protein level and specific
• enzyme activity


 De-gas :
 Gas will create bubbles that interfere movement of protein
from fractionized accordingly
i. Initial Buffer
ii. DE23
iii. (b) solution
iv.
• Glass wool is placed at the bottom of the glass column
• Glass column is slanted 45 and DE23 is poured until matrix
level reach 10-12cm
• Glass column is eluted with initial buffer (0.05M Tris pH7.3)
 Buffer will wash out contaminants present and separate
 charged proteins that are eluted out
• F3 is pipetted into glass column and let it absorbed by
matrix
•
• 20mL solution i is pipetted into the column
• Each 3mL fraction is collected into different test tubes
(done quickly)
• Above step is repeated for other solutions Higher
concentration of buffer weaker negative charge protein
to be eluted out
•
•
• By using quartz cuvette, absorbance is read at 280nm
•
•
• Graph of elution profile is plot to determine the protein
peak that shows an invertase enzyme activity
•
Results
Separation of Fraction III
by Ion Exchange
Chromatography
No. of test Group 4 & Group 5 & Group 15 & Group 11 & 21 0.216 0.002 0.010 0.010
tubes 9 13 17 1
1 0.048 0.015 0.069 0.069 22 0.019 0.004 0.009 0.009

2 0.021 0.014 0.041 0.041 23 0.357 0.008 0.006 0.006

3 0.018 0.068 0.358 0.381 24 0.314 0.002 0.023 0.019

4 0.306 0.029 0.041 0.041 25 0.349 0.033 0.008 0.008

5 0.013 0.002 0.014 0.014 26 0.333 0.001 0.010 0.011

6 0.038 0.003 0.003 0.005 27 0.339 0.001 0.005 0.005

7 0.035 0.007 0.041 0.041 28 0.749 0.001 0.001 0.001

8 0.039 0.002 0.042 0.042 29 0.302 0.002 0.007 0.007

9 0.036 0.010 0.066 0.066 30 0.022 0.001 0.022 0.020

10 0.239 0.020 0.090 0.090 31 0.033 0.004 0.003 0.003

11 0.044 0.009 0.103 0.103 32 0.016 0.000 0.035 0.033

12 0.207 0.011 0.119 0.120 33 0.058 0.007 0.006 0.006

13 0.027 0.005 0.090 0.091 34 0.058 0.000 0.002 0.002

14 0.033 0.018 0.139 0.150 35 0.043 0.005 0.001 0.001

15 0.039 0.007 0.081 0.074 36 0.188 0.000 0.005 0.005

16 0.028 0.001 0.061 0.083 37 0.000 0.003 0.019 0.016

17 0.026 0.003 0.061 0.061 38 0.000 0.000 0.009 0.009

18 0.103 0.002 0.062 0.062 39 0.000 0.000 0.008 0.007

19 0.040 0.010 0.051 0.051 40 0.000 0.003 0.006 0.006

41 0.000 0.001 0.003 0.005

20 0.027 0.004 0.007 0.007
42 0.000 0.003 0.004 0.004
Protein standard curve using Lowry
method
(from experiment 2)
Glucose standard curve using
Somogyi-Nelson Method
(from experiment 2)
 Estimation of Protein
amount by using Lowry
method
(for different fraction that is extracted
out from different concentration of salt)
 Absorbance of protein using
Lowry Method at 700nm
G ro u p 1 1 &1 1 5 &7 4 &9 5 &1 3
20m M 0 .1 0 3 0 .1 0 9 0 .0 4 5 0 .1 0 7
40m M 0 .0 6 8 0 .0 3 2 0 .0 3 4 0 .0 7 9
60m M 0 .0 5 7 0 .0 4 0 .0 5 9 0 .0 7 4
80m M 0 .0 4 3 0 .0 6 8 0 .0 7 5 0 .0 9 6
100m M 0 .0 3 3 0 .0 3 9 0 .0 2 7 0 .0 0 0
By using the following formula, protein amount is
calculated.

P ro te in a m o u n t( µ g )

G ro u p 1 1 &1 1 5 &7 4 &9 5 &1 3

20m M 103 109 45 107

40m M 68 32 34 79

60m M 57 40 59 74

80m M 43 68 75 96

100m M 33 39 27 0
Pro te in a m o u n t for different
fra ctio n in d iffe re n t g ro u p
Estimation of Enzyme
activity and Specific
enzyme activity by
using Somogyi-Nelson
method
(for diffent fraction that is extracted out
from different concentration of salt)
Absorbance of reducing sugar
using Somogyi-Nelson Method
at 510nm
G ro u p 1 1 &1 1 5 &7 4 &9 5 &1 3

20m M 0 .2 0 8 0 .1 1 7 0 .0 7 2 0 .0 0 0

40m M 0 .1 1 2 0 .0 6 4 0 .0 5 6 0 .0 6 8

60m M 0 .0 7 4 0 .0 8 3 0 .1 0 1 0 .1 4 1

80m M 0 .0 6 2 0 .0 9 4 0 .2 3 8 0 .0 8 2

100m M 0 .0 4 2 0 .0 8 0 .0 4 3 0 .0 0 0
By using the following formula, reducing sugar amount
is calculated.

re d u cin g su g a r a m o u n t( µ m o l)

G ro u p 1 1 &1 1 5 &7 4 &9 5 &1 3

20m M 1 .0 7 2 0 .6 0 3 0 .3 7 1 0 .0 0 0

40m M 0 .5 7 7 0 .3 3 0 0 .2 8 9 0 .3 5 1

60m M 0 .3 8 1 0 .4 2 8 0 .5 2 1 0 .7 2 7

80m M 0 .3 2 0 0 .4 8 5 1 .2 2 7 0 .4 2 3

100m M 0 .2 1 6 0 .4 1 2 0 .2 2 2 0 .0 0 0
B y u sin g th e fo llo w in g fo rm u la , e n zym e a ctivity is
ca lcu la te d .

E n zym e a ctivity ( µ m o l/ m in )

G ro u p 1 1 &1 1 5 &7 4 &9 5 &1 3

20m M 0 .1 0 7 0 .0 6 0 0 .0 3 7 0 .0 0 0

40m M 0 .0 5 8 0 .0 3 3 0 .0 2 9 0 .0 3 5

60m M 0 .0 3 8 0 .0 4 3 0 .0 5 2 0 .0 7 3

80m M 0 .0 3 2 0 .0 4 8 0 .1 2 3 0 .0 4 2

100m M 0 .0 2 2 0 .0 4 1 0 .0 2 2 0 .0 0 0
E n zym e A ctivity fo r d iffe re n t
fra ctio n in d iffe re n t g ro u p
B y u sin g th e fo llo w in g fo rm u la , sp e cific e n zym e a ctivity
is ca lcu la te d .

S p e cific E n zym e A ctivity ( µ m o l / m in m g )

G ro u p 1 1 &1 1 5 &7 4 &9 5 &1 3

20m M 1 .0 4 1 0 .5 5 3 0 .8 2 5 0 .0 0 0

40m M 0 .8 4 9 1 .0 3 1 0 .8 4 9 0 .4 4 4

60m M 0 .6 6 9 1 .0 7 0 0 .8 8 2 0 .9 8 2

80m M 0 .7 4 3 0 .7 1 3 1 .6 3 6 0 .4 4 0

100m M 0 .6 5 6 1 .0 5 7 0 .8 2 1 0 .0 0 0
Specific Enzyme Activity for
different fraction in different
group
 REFERENCES
• http://en.wikipedia.org/wiki/Carboxymethyl_
cellulose
• http://www.biology-
online.org/dictionary/Elution
• http://hplc.chem.shu.edu/NEW/HPLC_Book/R
ev.-Phase/rp_grad.html
• http://www.proteinchemist.com/tutorial/iec.h
tml
• http://en.wikipedia.org/wiki/Ion-
exchange_chromatography
• http://en.wikipedia.org/wiki/Ion_exchange