Sodium Dodecyl Poly Acryl AMide Gel electrophoresis (SDS-PAGE)

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SODIUM DODECYL SULPHATE POLY ACRYL AMIDE GEL ELECTROPHORESIS V. MAGENDIRA MANI ASSISTANT PROFESSOR PG & RESEARCH DEPARTMENT OF BIOCHEMISTRY ISLAMIAH COLLEGE (AUTONOMOUS) VANIYAMBADI [email protected] https://tvuni.academia.edu/mvinayagam

Transcript of Sodium Dodecyl Poly Acryl AMide Gel electrophoresis (SDS-PAGE)

Page 1: Sodium Dodecyl Poly Acryl AMide Gel electrophoresis (SDS-PAGE)

SODIUM DODECYL SULPHATE POLY

ACRYL AMIDE GEL ELECTROPHORESIS

V. MAGENDIRA MANI

ASSISTANT PROFESSOR

PG & RESEARCH DEPARTMENT OF BIOCHEMISTRY

ISLAMIAH COLLEGE (AUTONOMOUS)

VANIYAMBADI

[email protected]

 https://tvuni.academia.edu/mvinayagam  

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SODIUM DODECYL

SULPHATE POLY ACRYL

AMIDE GEL

ELECTROPHORESIS

 

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Principle

SDS PAGE is widely used method for separating

protein mixture and for determining the molecular weights.

SDS - is an anionic detergent

Binds strongly to protein

Causes their Denaturation

Polymerization

Cross linked poly acryl amide gel are formed by

polymerization of acryl amide monomer and N, N’ Methylene

Bisacryl amide in the presence of ammonium per sulphate and

TEMED (Tetra Methylene Diamine). Ammonium per sulphate

acts as free radical catalyst and TEMED acts as initiator.

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Photopolymerization

Photopolymerization is an alternative method that can be

used to polymerise Acryl amide gels. Ammonium per

sulphate, TEMED are replaced by Riboflavin. When the

gel is poured it is placed in front of bright light for 2 – 3

hrs. Photopolymerization of riboflavin generates a free

radical that initiates polymerization.

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Pore size of the PAGE

 Pore size of the gel can be varied by changing the

concentration of both Acryl amide and Methylene Bisacryl

amide. PAGE can be made with content of 3 % to 30 %. Low

percentage of gel 3 % have large pore size and used for

separation of proteins and DNA. High percentage of gel 10 -20

% have smaller pore size and used for SDS PAGE.

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Sample preparation

The sample is boiled with the buffer containing β-

mercaptoethanol and SDS for 5 min. β-mercaptoethanol

reduces any disulfide bridges in protein that are holding

tertiary structure of protein.

SDS binds strongly to proteins and denature the protein

On average one SDS molecule binds to every two amino

acids. So the native charge of the molecule is completely

swamped by SDS molecule (negative)

Each protein molecule will be fully denatured, opens in to

a rod shaped structure with a series of – ve charged SDS

molecule along with polypeptide chain.

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Gel preparation Gels used are vertical slabs, because it is more economical and

more sample can be compared with each other when run under

identical conditions (eg. 20 different samples)

Gels are prepared in glass containers in which they are to be

used. The two glass plates are held together but held apart from

each other by plastic spacer, vertical slab gels are run along with

the glass plate.

Choice of percentage of gel to be used depends on the size of

the protein sample. Separating gel used may vary from 10 %

PAGE to 15 % PAGE. 15 % of gel used for separation of protein

having molecular weight 10,000 to 1, 00,000 and 10 % of gel

used for separation of protein having molecular weight 1,

50,000.

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Sample application

Dissolved samples can be applied using a micro syringe into wells

of the gel. 

Sample buffer containing 10-15 % Sucrose or Glycerol, which

increase the density of the buffer and ensures the sinking of the

sample in to the wells.

 It also prevents the sample from mixing with buffer in the upper

buffer reservoir.

 Sample buffer contain marker/tracker dye Bromophenol blue. It

is a small molecule and it moves freely and indicates the

electrophoretic migration.

 Urea, SDS, Disulfide reducing agent such as β-Mercaptoethanol

are added to protein sample to facilitate their solubilisation.

 Protein sample can be loaded in the form of sharp band by using

a staking gel over the separating gel.

 Only µ g of sample are used for analyzing.

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Running the gel

 

The gel slab sandwiched in between the glass plate is placed in

the lower reservoir with the top of the gel in contact with the

buffer in the upper reservoir.

Thus the gel completes the electrical circuit between the lower

and upper compartments. Although the buffer dissipates the heat

generated, additional cooling may be needed.

In sample small protein can more easily pass through the pores

and larger proteins are successively retarded by frictional

resistance due to sieving effect of the gel.

Precise voltage and time required for the optimal separation

Voltage: 30 mA; Time; 3 hrs

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Detection

 When tracker dye reaches the bottom of the gel the current is

turned off.

 Gel slabs are removed without any pressure, after removal

gel is immersed in 7 % acetic acid to minimize diffusion of

components.

 Then the gel is shaken well in an appropriate stain solution.

Usually Commassive Brilliant Blue R250 is used and the gel

is immersed for few hours.

 Then the gel is transferred in to a destain solution and kept

for overnight.

 Destain solution removes unbound background dye from gel

leaving stain protein visible as blue bands on a clear

background.

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Staining solution (100 ml)

a. Coomassie Brilliant Blue R-250: 250 mg

b. Methanol: 50 %

c. Acetic acid: 10 %

d. Distilled water: 40 %

De-staining solution (100 ml)

a. Methanol: 50 %

b. Acetic acid: 10 %

c. Distilled water: 40 %

Time required for electrophoresis

Gel preparation - 1– 1 ½ hrs

Running the gel - 3 hrs

Staining - 2– 3 hrs

De-staining - overnight

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Determination of Molecular weight (PROTEIN) by SDS

PAGE.

 The Molecular weight can be determined comparing mobility

of standard protein of known Molecular weight with of

unknown Molecular weight that is run on the same gel.

A calibration curve is constructed for standard protein of

known Molecular weight by

Distance migrated Vs Molecular weight x 104

The migration of unknown is measured are extrapolating this

value in the calibration curve, the molecular weight of

unknown can be determined.

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V. MAGENDIRA MANI

ASSISTANT PROFESSOR

PG & RESEARCH DEPARTMENT OF BIOCHEMISTRY

ISLAMIAH COLLEGE (AUTONOMOUS)

VANIYAMBADI

[email protected]

 https://tvuni.academia.edu/mvinayagam