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Flussdiagrammknoten

• 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