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Separation of serum protein on SDS-PAGE

Set up the apparatus as in figure 8 to pour a gel , using the casting stand and spacers of 1.5 mm

Prepare the running gel in 100 ml beaker

Pour the freshly mixed running gel in to space between two parallel glass plates in the casting stand

Add water layer above the running gel , allow the gel to polymerize for at least 30 min , then pour of the water from the polymerized running gel

Prepare the stacking gel

Pour the stacking gel in to the running gel

17.8 ml distilled water

13.6 ml 30% acrylamide solution

8 ml 1.875 M Tri-HCl , pH 8.8

400 Ml 10% SDS

20 Ml TEMED

200 Ml freshly dissolved ammonium persulfate

3.75 ml distilled water

0.68 ml 30% acrylamide solution

0.5 ml 0.6 M Tris-HCl ,pH 8.8

50 10% SDS

5 TEMED

25 freshly dissolved ammonium persulfate

Insert the comb to create sample wells in the stacking gel , allow the gel to polymerize to 30 min at RT

Place thetus polymerized gel in to the plates into the electrophoresis appara

Add running buffer to the chambers at the bottom and top of the gel , remove the comb from between the plates

Prepare the standard and unknown protein samples

mix 10 Ml of each with 30 Ml of sample buffer , mix and heat at 100 C for 5 min

Load the samples onto the gel with a syringe

Connect the electrode of the apparatus to the power source

Apply a 50 mamp current , 200 V to the apparatus and continue electrophoresis until the bromophenol blue reach the bottom of the gel

Turn off the power and disconnect the electrodes , remove the gel from apparatus , by sepatula , separat the two plates and separate the soft stacking gel from the more rigid running gel

Transfer the running gel into pan filled with 50 ml of coomassie blue solution allow it to soak for 30 min

Pour off the staining solution and replace it with destaining solution for one hour , replacing it with fresh destainung solution every 15 min

Destaining is complete when the protein appaer as blue bands on transparent gel