Health Information

Coomassie brilliant blue Staining

This is the most often used protein stain. It is extremely sensitive and becomes even more sensitive in the presence of sodium dodecyl sulphate. It can be carried out by two variants of what is principally one single method.

The first step is to fix the separated proteins. This is achieved by soaking the gels overnight in about 10-12 volumes of 20% sulphasalicyclic acid. This step is followed by staining where the gels are allowed to soak in an aqueous solution of Coomassie blue (0.25%). There is no fixed period for staining. However, higher percentage gels require higher periods of staining. Thus, while 2 hours are sufficient for 5% acrylamide gels, more than 4 hours are required for staining 10% acrylamide gels. Excess stain is removed by washing the gel with successive volumes of 7% acetic acid.

Overnight fixation of proteins is quite time consuming. This problem is obviated by combining the fixing and steps. This is achieved by soaking the gels in a Coomassie brilliant blue solution in methanol and glacial acetic acid. The period of soaking might vary from 2-10 hours. Excess stain is removed by washing the gels in a solution of glacial acetic acid, methanol and water. This method consumes significantly less time and is consequently very widely used.

Fluorescent staining

There are two good fluorescent stains for proteins. The first is fluorescamine; this compound is non-fluorescent in the uncomplexed state. However, it can react very specifically with primary amines, e.g., lysine, and upon such a reaction the complex formed is fluorescent. The reaction and therefore the staining takes a very short time and on this count the method scores more points than the Coomassie blue staining which is time consuming. Fluorescamine is applied to proteins before the electrophoresis if SDS –containing gels are to be used.

The second fluorescent stain, which needs mention, is anilinonapthalene sulphonate (ANS) which does not fluoresce in water. It does, however, fluoresce when dissolve in organic solvents or when it becomes bound to the hydrophobic regions of proteins.