In-gel Digestion - Destaining

Destaining

Proteins which were separated by 1D or 2D PAGE are usually visualised by staining with dyes like Coomassie Brilliant Blue (CBB) or silver. Although the sensitivity of the method is significantly lower, the use of Coomassie is more common for samples destined for mass spectrometry since the silver staining impairs the analysis. After excision of the protein band of interest from the gel most protocols require a destaining of the proteins before proceeding.

The destaining solution for CBB contains usually the buffer salt ammonium bicarbonate (NH₄HCO₃) and a fraction of 30%-50% organic solvent (mostly acetonitrile). The hydrophobic interactions between protein and CBB are reduced by the organic fraction of the solution. At the same time diminishes the ionic part of the solution the electrostatic bonds between the dye and the positively charged amino acids of the protein. In contrast to a mixture of water with organic solvent the effectivity of destaining is increased. An increase of temperature promotes the destaining process. To a certain degree (< 10%) the destaining procedure is accompanied with a loss of protein. Furthermore, the removal of CBB does not affect the yield of peptides in the mass spectrometric measurement.

In the case of silver stained protein bands the destaining is accomplished by oxidation of the metallic silver attached to the protein by potassium ferricyanide or hydrogen peroxide (H₂O₂). The released silver ions are complexed subsequently by sodium thiosulfate.