Figure 35.4: Different steps in performace of vertical gel electrophoresis to resolve sample.

Running of the gel: The sample is prepared in the loading dye containing SDS, β-mercaptoethanol in glycerol to denature the sample and presence of glycerol facilitates the loading of sample in the well. As the samples are filled vertically there is a distance drift between the molecules at the top Vs at the bottom in a lane. This problem is taken care once the sample run through the stacking gel. The pH of the stacking gel is 6.8 and at this pH, glycine is moving slowly in the front where as Tris-HCl is moving fast. As a result, the sample gets sandwiched between glycine-Tris and get stacked in the form of thin band. As the sample enters into the resolving gel with a pH 8.8, the glycine is now charged, it moves fast and now sample runs as per their molecular weight (due to SDS they have equal negative charge). After tracking dye reaches to the bottom of the gel, gel is taken out from the glass plate with the help of a spatula and it is stained with coomassie brilliant blue R250 dye. The dye stains protein present on the gel.   A typical SDS-PAGE is given in the Figure 35.5.