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10.3.7 Detection, estimation and recovery of proteins in gels

The most commonly used general protein stain for detecting protein on gels is the

sulphated trimethylamine dye Coomassie Brilliant Blue R-250 (CBB). Staining is

usually carried out using 0.1% (w/v) CBB in methanol:water:glacial acetic acid

(45:45:10, by vol.). This acid–methanol mixture acts as a denaturant to precipitate

or fix the protein in the gel, which prevents the protein from being washed out whilst

it is being stained. Staining of most gels is accomplished in about 2 h and destaining,

usually overnight, is achieved by gentle agitation in the same acid–methanol solution

but in the absence of the dye. The Coomassie stain is highly sensitive; a very weakly

staining band on a polyacrylamide gel would correspond to about 0.1mg (100 ng) of

protein. The CBB stain is not used for staining cellulose acetate (or indeed protein

blots) because it binds quite strongly to the paper. In this case, proteins are first

denatured by brief immersion of the strip in 10% (v/v) trichloroacetic acid, and then

immersed in a solution of a dye that does not stain the support material, for example

Procion blue, Amido black or Procion S.

Although the Coomassie stain is highly sensitive, many workers require greater

sensitivity such as that provided by silver staining. Silver stains are based either on

techniques developed for histology or on methods based on the photographic process.

In either case, silver ions (Agþ) are reduced to metallic silver on the protein, where the

silver is deposited to give a black or brown band. Silver stains can be used immedi-

ately after electrophoresis, or, alternatively, after staining with CBB. With the latter

Albumin

Sample application

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IgG

Fig. 10.10Electrophoresis of human serum samples on an agarose gel. Tracks 2, 3, 4 and 6 show normal serum

protein profiles. Tracks 1, 5 and 7 show myeloma patients, who are identified by the excessive production of a

particular monoclonal antibody seen in the IgG fraction. (Courtesy of Charles Andrews and Nicholas Cundy,

Edgware General Hospital, London.)

417 10.3 Electrophoresis of proteins