Amali 5 to Present

8/8/2019 Amali 5 to Present

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OBJECTIVE

the process of preparing matrix DEAE-Cellulose

To prepare the elution buffer

To separate fractio

n III (Invertase) through ion exchange

.hnique of taking the absorbance using quartz cuvete


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INTRODUCTIO

N


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ION EXCHANGECHROMATOGRAPHY

Process of separation of ions andpolar molecules based on theircharge.

Eg : large proteins, small nucleotides,and amino acids.

Usually used in protein purification,

water analysis and quality control.


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ION EXCHANGE

Exchange of ions between twoelectrolytes.

Mostly used to denote the purification,

and separation process. Reversible process.

Ion exchanger : cations / anions /amphoteric

Anion exchangers exchange negativelycharged ions.

Cations exchangers exchange the

positively charged ions.


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ION EXCHANGE

Amphoteric exchangers, are able toexchange both cations and anionssimultaneously.However, the

simultaneous exchange of cations andanions can be more efficientlyperformed in mixed beds that containa mixture of anion and cationexchange resins.

Ions exchanger are able to beregenerated / loaded with desirable

ions by washing with an excess of


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PRINCIPLES OF ION EXCHANGECHROMATOGRAPHY

Relies on charge-charge interactionsbetween the proteins in the sampleand 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 anegatively charged resin

- Anion exchange chromatography ;the binding ions are negative, and the


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PRINCIPLES OF ION EXCHANGECHROMATOGRAPHY

If you know the pH you want to run at andneed to decide what type of ion exchangeto use paste your protein sequence intothe 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 theprotein will flow through while the

contaminants will bind. flow throughmode


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MATERIALS

- ( )Diethylaminoethyl cellulose DE 23 . ( )0 50M Hydrochloric acid HCl . ( )0 50m Sodium hydroxide NaOH

.0 05M Tris buffer ( )Sodium Chloride NaCl


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PREPARATION OF TRISBUFFER

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


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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


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= 4.927 10 -2 kg 1.19 kg 1L

= 0.0414 L

= 41.4 ml

Top up to 1 L


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PREPARATION OF NaCl

vn= MV = (200 10) 0.50 L = 0.1 mol

vMass = 0.1 58.5 = 5.85 g

Top up to 500 ml


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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 mliii. 60mM 50 ml = 200 V2 V2= 15 ml


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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


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A) Preparation of DEAE-

cellulose matrix10g dry DE23-weight

200ml (0.50M)HCI added

Stir for 1 hour

Let matrix precipitated


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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


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Let matrix precipitated

Water was removed

Wash DE23 matrix until ph water 8.0 DE23 matrix-keep in distilled

water


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B)Preparation of elutionbuffer

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 obtainbuffer 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)


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C) Separation of F3 by ion exchangechromatography

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 Bufferii. 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 matrixlevel 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


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F3 is pipetted into glass column and let it absorbed bymatrix

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 proteinto be eluted out

By using quartz cuvette, absorbance is read at 280nm

Graph of elution profile is plot to determine the proteinpeak that shows an invertase enzyme activity


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Results


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Separation of Fraction IIIby Ion ExchangeChromatography


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.No of testtubes

&Group 49

&Group 513

&Group 1517

&Group 111

1 .0 048 .0 015 .0 069 .0 069

2 .0 021 .0 014 .0 041 .0 041

3 .0 018 .0 068 .0 358 .0 381

4 .0 306 .0 029 .0 041 .0 041

5 .0 013 .0 002 .0 014 .0 014

6 .0 038 .0 003 .0 003 .0 005

7 .0 035 .0 007 .0 041 .0 041

8 .0 039 .0 002 .0 042 .0 042

9 .0 036 .0 010 .0 066 .0 066

10 .0 239 .0 020 .0 090 .0 090

11 .0 044 .0 009 .0 103 .0 103

12 .0 207 .0 011 .0 119 .0 120

13 .0 027 .0 005 .0 090 .0 091

14 .0 033 .0 018 .0 139 .0 150

15 .0 039 .0 007 .0 081 .0 074

16 .0 028 .0 001 .0 061 .0 08317 .0 026 .0 003 .0 061 .0 061

18 .0 103 .0 002 .0 062 .0 062

19 .0 040 .0 010 .0 051 .0 051

20 .0 027 .0 004 .0 007 .0 007

21 .0 216 .0 002 .0 010 .0 010

22 .0 019 .0 004 .0 009 .0 009

23 .0 357 .0 008 .0 006 .0 006

24 .0 314 .0 002 .0 023 .0 019

25 .0 349 .0 033 .0 008 .0 008

26 .0 333 .0 001 .0 010 .0 011

27 .0 339 .0 001 .0 005 .0 005

28 .0 749 .0 001 .0 001 .0 001

29 .0 302 .0 002 .0 007 .0 007

30 .0 022 .0 001 .0 022 .0 020

31 .0 033 .0 004 .0 003 .0 003

32 .0 016 .0 000 .0 035 .0 033

33 .0 058 .0 007 .0 006 .0 006

34 .0 058 .0 000 .0 002 .0 002

35 .0 043 .0 005 .0 001 .0 001

36 .0 188 .0 000 .0 005 .0 005

37.

0 000.

0 003.

0 019.

0 016

38 .0 000 .0 000 .0 009 .0 009

39 .0 000 .0 000 .0 008 .0 007

40 .0 000 .0 003 .0 006 .0 006

41 .0 000 .0 001 .0 003 .0 005

42 .0 000 .0 003 .0 004 .0 004


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Protein standard curve using Lowry

method(from experiment 2)

ucose s an ar curve us ng


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ucose s an ar curve us ngSomogyi-Nelson Method

(from experiment 2)


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Estimation of Proteinamount by using Lowry

method(for different fraction that is extracted

out from different concentration of salt)


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Absorbance of protein usingLowry Method at 700nm

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

2 0 m M .0 1 0 3 .0 1 0 9 .0 0 4 5 .0 1 0 7

4 0 m M .0 0 6 8 .0 0 3 2 .0 0 3 4 .0 0 7 9

6 0 m M .0 0 5 7 .0 0 4 .0 0 5 9 .0 0 7 4

8 0 m M .0 0 4 3 .0 0 6 8 .0 0 7 5 .0 0 9 6

1 0 0 m M .0 0 3 3 .0 0 3 9 .0 0 2 7 .0 0 0 0


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By using the following formula, protein amount iscalculated.

( )P ro te in a m o u n t g

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

2 0 m M 1 0 3 1 0 9 4 5 1 0 7

4 0 m M 6 8 3 2 3 4 7 9

6 0 m M 5 7 4 0 5 9 7 4

8 0 m M 4 3 6 8 7 5 9 6

1 0 0 m M 3 3 3 9 2 7 0


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Pro te in a m o u n tfor differentfra ctio n in d iffe re n t g ro u p


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Estimation of Enzymeactivity and Specificenzyme activity by

using Somogyi-Nelsonmethod

(for diffent fraction that is extracted outfrom different concentration of salt)

Absorbance of reducing sugar


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Absorbance of reducing sugarusing Somogyi-Nelson Method

at 510nmG ro u p &1 1 1 &1 5 7 &4 9 &5 1 3

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

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

6 0 m M .0 0 7 4 .0 0 8 3 .0 1 0 1 .0 1 4 1

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

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

i h f ll i f l d i


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By using the following formula, reducing sugar amountis calculated.

( )red u cin g su g ar am ou n t m ol

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

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

4 0 m M .0 5 7 7 .0 3 3 0 .0 2 8 9 .0 3 5 1

6 0 m M .0 3 8 1 .0 4 2 8 .0 5 2 1 .0 7 2 7

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

1 0 0 m M .0 2 1 6 .0 4 1 2 .0 2 2 2 .0 0 0 0

B i h f ll i f l i i i


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,B y u sin g th e fo llo w in g fo rm u la e n zy m e a ctiv ity is.ca lcu la te d

( / )E n zym e a ctiv ity m o l m in

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

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

4 0 m M .0 0 5 8 .0 0 3 3 .0 0 2 9 .0 0 3 5

6 0 m M .0 0 3 8 .0 0 4 3 .0 0 5 2 .0 0 7 3

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

1 0 0 m M .0 0 2 2 .0 0 4 1 .0 0 2 2 .0 0 0 0

f d ff


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E n zy m e A ctiv ity fo r d iffe re n tfra ctio n in d iffe re n t g ro u p

B i th f ll i f l ifi ti it


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,B y u sin g th e fo llo w in g fo rm u la sp e cific e n zy m e a ctiv ity.is ca lcu la te d

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

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

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

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

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

8 0 m M .0 7 4 3 .0 7 1 3 .1 6 3 6 .0 4 4 0

1 0 0 m M .0 6 5 6 .1 0 5 7 .0 8 2 1 .0 0 0 0

Specific Enzyme Activity for


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Specific Enzyme Activity fordifferent fraction in different

group


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REFERENCES

http://en.wikipedia.org/wiki/Carboxymethyl_cellulose

http://www.biology-online.org/dictionary/Elution

http://hplc.chem.shu.edu/NEW/HPLC_Book/Rev.-Phase/rp_grad.html

http://www.proteinchemist.com/tutorial/iec.html

http://en.wikipedia.org/wiki/Ion-exchange_chromatography

http://en.wikipedia.org/wiki/Ion_exchange

Amali 5 to Present

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