Biochemical Techniques and Instrumentation

Procedure

1. Thoroughly clean and dry the glass plates and spacers. Assemble them properly. Hold the construction together with bulldog clips, clamps in upright level position. White petroleum jelly or 2% agar (melted in a boiling waterbath) is then applied around the edges of the spacers to hold them in place and seal the chamber between the glass plates.
2. Prepare sufficient volume of separating gel mixture by mixing the following:

Reagents	15% gel	12.5 % gel	10% gel
Stock acrylamide solution	20.1 ml	16.7 ml	13.3 ml
Tris – HCl (pH 8.8)	8.0 ml	8.0 ml	8.0 ml
Water	11.4 ml	14.7 ml	18.2 ml
Degas in a water pump for 3-4 min and then add
Ammonium persulfate solution	0.2 ml	0.2 ml	0.2 ml
10% SDS	0.4 ml	0.4 ml	0.4 ml
TEMED	20 m l	20 m l	20 m l

3. Mix gently and carefully pour the gel solution in the chamber between the glass plates. Layer distilled water on top of the gel and leave to set for 30-60 min
4. Prepare stacking gel (1%) by mixing the following solutions (total volume 10 ml)

Reagents	10 ml
Stock acrylamide solution	1.35 ml
Tris – HCl (pH 6.8)	1.0 ml
Water	7.5 ml
Degas in a water pump for 3-4 min and then add
Ammonium persulfate solution	50 m l
10% SDS	0.1 ml
TEMED	10 m l

Remove the water from the top of the gel and wash with a little stacking gel solution. Pour the stacking gel mixture, place the comb in the stacking gel and allow the gel to set for (30-60 min)

5. After the stacking gel has polymerized, remove the comb without distorting the shapes of the well. Carefully install the gel in the electrophoresis apparatus. Fill it with electrode buffer and remove any trapped air bubbles at the bottom of the gel. Connect the cathode at the top (the stacking gel end of the gel) and turn on the DC power briefly to check the electrical circuit.
6. Prior to or during gel polymerization prepare samples for electrophoresis following suitable extraction procedure. In case of fish, take 2 g of fish, homogenize with 10 ml of buffer¸ centrifuge and collect the supernatant. Adjust the protein concentration in each sample using the 5 X sample buffer and water in such a way that the same amount of protein is present per unit volume. The concentration should be such as to give a sufficient amount of protein (50-200 m g) in a volume (25-50 m l) not greater than the size of the sample well. As a general practice, heat sample solutions in boiling water for 2-3 min, to ensure complete reaction between proteins and SDS.
7. Cool the sample solution and load in the gel using a micropipette. Mark the position of wells on the glass plate with a marker pen and the presence of bromophenol blue in the sample buffer facilitate easy loading. Similarly load a few wells with standard marker proteins in the sample buffer.
8. Turn on the current to 10-15 mA for initial 10-15 min, until the samples travel through the stacking gel. This helps in concentration of the samples. Then continue the run at 100 mA until the bromophenol blue reaches the bottom of the gels..
9. After the run is complete, carefully remove the gel from between the plates and immerse in the staining solution for at least 3 h or overnight with uniform shaking. The proteins absorb the coomassic brilliant blue.
10. Transfer the gel to a suitable container with at least 200-300 ml destaining solution and shake gently, continuously. Dye that is not bound to proteins is thus removed. Change the destaining solution frequently, particularly during initial periods, until the background of the gel is colourless. The proteins fractionated into band are seen colored blue. As the proteins of minute quantities are stained faintly, destaining process should be stopped at appropriate stage to visualize as many bands as possible.