SDS PAGE PROTOCOL

INTRODUCTION
SDS-PAGE is the most widely used technique to separate proteins in various samples of mixture. In poly-acrylamide gel proteins migerate to the negative anode under denaturing conditions. In SDS-PAGE, the detergent SDS and a heating step result that the electrophoretic mobility is only determined by molecular weight of proteins. An intact protein electrophoresis system containing: a tank, lid with power cables, electrode assembly, cell buffer dam, casting stands, casting frames, combs(usually 10-well or 15-well), and glass plates (thickness 0.75mm or 1.0mm or 1.5mm). Protein gels in a single electrophoresis running are divided into stacking gel and separating gel. Stacking gel (acrylamide 5%) is poured on top of the separating gel and a gel comb is inserted in the stacking gel. The acrylamide percentage in the separating gel depends on the size of the target protein in the sample.

MATERIALS
To Pour Gels: 30% acrylamide 10% SDS 10% APS (make fresh each time) TEMED 1.5 M Tris, pH 8.8 (resolving gel) M Tris, pH 6.8 (stacking gel)

5x SDS Running Buffer (1 L) Tris 15 g Glycine 72 g SDS 5 g

Coomassie Blue Stain 10% (v/v) acetic acid 0.006% (w/v) Coomassie Blue dye 90% ddH2O

Isopropanol Fixing Solution 10% (v/v) acetic acid 25% (v/v) isopropanol 65% ddH2O

SDS sample loading buffer (40 ml) ddH2O 16 ml 0.5 M Tris, pH 6.8 5 ml 50% Glycerol 8 ml 10% SDS 8 ml 2-mercaptoethanol 2 ml (add immediately before use) bromophenol blue

10% (v/v) acetic acid

METHODS
1. Make the gel: Set the casting frames (clamp two glass plates in the casting frames) on the casting stands. Pour appropriate amount of separating gel solution into the gap between the glass plates. Then pour water (we use isopropanol sometimes) into the gap untill a overflow. Wait for 20-30min to let it condense. Pour out the water and you can see separating gel left. Then pour stacking gel untill a overflow. Then insert the comb. Wait for 20-30min to let it condense. 2. Make sure a complete condensation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer into the inner chamber untill the buffer surface reaches the required level in the outer chamber. 3. Prepare the samples: Mix your samples with sample buffer. Heat them in boiling water for 5-10 min. 4. Load them into the well. Don't forget loading protein marker into the first lane. Then cover the top and connect the anodes. 5. Choose an appropriate volt and run the electrophoresis when everything's ok. 6. Typically, about 1 hour for a 120V voltage and a 12% separating gel. For a higher percentage of acylamide, the time will be longer.

7. Remove the gel for the power supply and process further - Visualize your proteins using Coomassie Brilliant Blue, Silver stain, or any of the other protein stains. Use a carbohydrate stain for glycoproteins, or blot your gel for N-terminal sequencing or Western blotting

OBSERVATION AND ANALYSIS

For the observation and analysis of the proteins separated during SDS-Page, an entirely distinct molecular technique is used.Immunoblotting, also known as western blotting, involves the detection of specific proteins by antibodies. Proteins that have been separated by SDS-PAGE can then transferred electrophoretically to a membrane. This is necessary because staining proteins in the gel using antibodies is impractical due to poor penetrations of the antibodies into the gel. The most commonly used membranes are composed of nitrocellulose, and most proteins will bind tightly to nitrocellulose, thus becoming immobilized. The transfer is accomplished by placing the nitrocellulose membrane directly on the gel in a sandwich, with the gel and membrane pressed together between layers of porous padding and filter paper. This sandwich is then placed into an electrophoresis rig in a suitable buffer, and power is applied cross-wise to the gel-membrane sandwich, with the membrane toward the positive terminal. Since the proteins retain their negative charge from SDS-PAGE, they will migrate out of the gel onto the nitrocellulose membrane and become firmly bound to it. After electrophoretic transfer, in anticipation of exposure to antibodies, the nitrocellulose membrane is soaked in a protein-containing solution such as non-fat milk or bovine serum albumin (BSA) to encourage the formation of non-specific protein interactions. This blocking step is necessary to prevent the antibodies (which are also proteins) from forming the same type of non-specific interactions in subsequent steps of the procedure. Also, the blocking proteins will prevent the antibodies from binding over the entire surface of the nitrocellulose, which would happen otherwise.The blocking solution is then washed off the membrane, and the membrane is then exposed to an antibody (the primary antibody). The primary antibody interacts specifically and with high affinity with its antigen, and will displace proteins from the blocking solution that interact nonspecifically. Following exposure to the primary antibody, the membrane is washed to remove excess antibody, and then exposed to a second antibody (the secondary antibody) which binds to the primary antibody. The secondary antibody is covalently attached to an enzyme, such as alkaline phosphatase or horseradish peroxidase, which can be used to generate a colored product from a colorless reaction mixture. The product precipitates on the nitrocellulose membrane at the location of enzyme (and the antibody to which it is attached). Thus the presence of the antigen on the membrane can be detected and specifically distinguished from a host of other proteins in solution

The colored product and the intensity of the color give us vital clues which help detect and single out the proteins that were separated during SDS-Page.