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SDS-Page Analysis of Proteins

1. Objective:

Analysis of protein by gel eletrophoressis under denaturing conditions (SDS – PAGE)

2. Introduction :

The proteins, DNA and RNA (biomolecules) have electric charges which depend on molecule to molecule and the conditions of the medium (pH of buffer in which dissolved). Charged molecules can be separated by electrophoresis in gels.

Polyacrylamide gel is mainly used for protein isolation. Due to the differences in amino acid composition proteins have a unique mass and charge. Hence, the proteins have net negative charge and net positive charge or isoelectric point (no charge) given pW of buffer.

SDS – PAGE is a high resolution method used universally for analysing the mixture proteins according to their respective size. SDS solubilised in soluble proteins makes possible analysis of the other insoluble mixtures.

Separation of the proteins does not occur due to s charge: mass ratio (z/m). Therefore, such proteins are treated first with an ionic detergent sodium dodecyl sulphate (SDS) before the start and during the course of electrophoresis (P. Therefore, such electrophoresis is called SDS – PAGE.

Identical proteins are denatured by SDS resulting in their sub-units. The polypeptide get opened and extended.

On the basis of their mass but not the charge, the molecules are sep Electrophoretic separation is normally used for these reasons i.e. (i) gel acts as molecular s hence separates the molecules on the basis of their size, and (ii) gel suppresses conventionale produced by small temperature gradient which improves the resolution.

Polyacrylamide (supporting media) is used for this purpose due to its good nature (chemically inert, stable ov wide range of pW, temperature, ionic strength and transparent). Polyacrylamide gel is better size fraction of proteins.

SDS- PAGE by Laemmli Procedure:

Laemmli invented the disc gel electrophoresis is a superior system to resolve proteins in a mixture. According to the procedure proteins electrophoresed by a discontinuous polyacryamide gel.

A stacking gel (1/3 of total gel) w includes the sample loading wells consists of 4% acrylamide prepared in Tris HC1 buffer (pH6.8). The remaining 2/3 of the gel length is called separating gel.

The separating gel consists of 7.5- 15.0 % acrylamide. This range depends upon the molecular size of proteins prepared in Tris HCL buffer (pH 8.8). In analysis of a complex mixture of proteins the resolution is improved by 4 initial movements through a stacking gel.

The final bands in the separating gel are sharper and focused in a better way. After electrophoresis a mixture of proteins is separated as discrete bam (Fig. 12.2).Mixture of proteins before electrophoresis

3. Principle :

The proteins are denatured and have a negative charge with a uniform charge to mass ratio (z/m) when treated with SDS (anioic detergent). Proteins migrate toward anode at alkaline pH through PAGE gel during electrophoresis. The smaller polypeptides moves faster followed by the larger polypeptides.

Therefore, the intrinsic charge on proteins is masked in SDS-PAGE. Hen the separation is based on the size. Mercapto-ethanol reduces interpolypeptide disulfide – separates the sub-units of a polymeric protein.

4. Requirements :

i. Ammonium per sulphate (10 %), Coomassie brilliant blue (0.3 %)

ii. De-staining mixture, gel staining dish

iii. Electrophoresis apparatus with supply

iv. Running buffer, SDS (10 %), 1.5 M Tris HC1 (pH 8.8), 0.5 M Tris HC1 (pH 6.8), Laemmli buffer

v. Stock acrylamide – bis acrylamide solution

vi. Reagents: The following reagents must be prepared in advance

vii. Stock acrylamide (bis acrylamide solution: 29.2 g acrylamide, 0.8 g bis acrylaminde; final volume raised to 100 ml)

viii.1.5 M tris-HCI, pH 8.8: Dissolve 18.15 g of Tris in 50 ml of distilled water, adjust the pH to 8.8 with HC1, make the final volume to 100 ml.

ix. 0.5 M Tris- HCI, pH 6.8: Dissolve 6 g Tris in 60 ml distilled water, adjust pH to 6.8 with HC1, make the final volume to 100 ml.

x. 10% SDS: Dissolve lg SDS in 5 ml distilled water and raise the final volume equal to 10 ml.

xi. Gel running buffer: Dissolve 14.4 g glycine, 1 g SDS in 1 litre distilled water, adjust pW to 8.3 by adding solid Tris, make the final volume equal to 1 litre.

xii. Ammonium persulphate (APS) (10%): Dissolve 500 mg of solid APS’in 5 ml of distilled water. Always use freshly prepared APS solution only.

xiii.Coomassie Brilliant Blue R 250: Dissolve 600 mg of Coomassie brilliant Blue R 250 (CBBR-250) in 80 ml methanol, add 20 ml glacial acetic acid and make the final volume equal to 200 ml with distilled water.

xiv.De-staining solution: Mix 400 ml methanol, 100 ml glacial acetic acid and 500 ml distilled to get 1 litre of this solution.

xv. Laemmli buffer: 62.5 mM Tris-HCI, pU 6.8 (use diluted 0.5M Tris HCI pH 6.8), 10% glycerol, 5% mercapto-ethanol, 2% SDS.

According to the manufacturer’s instructions, gel electrophoesis apparatus should be assembled. Protein sample should be prepared in Laemmli buffer or in the buffer supplied with the kit for the experiment.

5. Procedure :

(i) Make 7.5% uniform concentration of SDS resolving gel. The capacity of the apparatus governs the volume of the final gel solution. Accordingly, read carefully the instruction manual supplied with the electrophoresis unit before deciding the volume of the gel. Commercially available SDS-PAGE kit is also used according to the instruction.

(ii) According to manufacturer’s instruction, assemble the gel electrophoresis apparatus.

(iii) Make gel solution of the separating and stacking gels (find out the volume of solution needed by the apparatus). Prepare fresh solution just before use following the Table 12.1.

APS and TEMED are added to the rest of the solution just before pouring the gel solution into the glass sandwich.

(iv) Fill up the glass sandwich with pipette upto mark with the separating gel solution prepare (the body must not make contact with chemicals. The acrylamide solution in unpolymerized state is neuro-toxic).

(v) To make the gel surface straight after polymerization, overlay the acrylamide solution with water (the gel should be allowed to polymerize for 30 minutes. When the polyacrylamide layer becomes distinct below the water layer, polymerization is complete).

(vi) Remove the distilled water by using the filter paper carefully. Thereafter, add the requisite amount of 4% stacking gel solution (freshly mixed). Then insert the comb for polymerization.

(vii) With the polymerized gel place the glass sandwich into the electro phoretic chamber I and add running buffer to level in both cathodic and anodic chambers. To prevent the sample wells from deforming, remove the comb under buffer.

(viii) With the help of micro-pipette, load the denatured protein solution into the well (protein sample should be denatured in Laemmli buffer by boiling for 5 minutes).

(ix) Add standard molecular weight marker proteins in one lane. For detection by CBB dye, generally 20 to 50 pg protein is sufficient.

(x) Tightly connecting the electrodes of the apparatus with the power supply. Run the gel at constant current of 20m A.

(xi) Track the mobility of sample in the matrix with the dye (generally bromophenol blue is added to the Laemmli buffer). After completion, switch off the button and disconnect the apparatus.

(xii) Transfer the gel to the staining tray containing the gel staining dye.

(xiii) Under shaking conditions on a rocking shaker, stain the gel for at least 2 hours or overnight. At this stage the whole gel turns blue.

(xiv) Carefully transfer the gel de-staining solution and shake on a rocker shaker for 30 minutes. Add fresh destaining solution. Repeat these steps until the bands are clearly visible in the gel. At this stage take photograph of the gel.

(xv) When the gel is clearly visible, analyze the photographed gel.

6. Results

(i) Several distinct blue coloured bands can be seen in the gel.

(ii) Each band represents to a single or multiple bands in the lane.

(iii) Depending on the amount of the polypeptide present in the protein solution loaded in the gel, the intensity of these bands varies.