The most commonly used general protein stain for detecting protein on gels is the sulfated 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 volume). This acid–methanol mixture acts as a denaturant to precipitate or fi x 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 0.1 μg (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 tech niques developed for histology or on methods based on the photographic process. In either case, silver ions (Ag +) are reduced to metallic silver on the protein, where the silver is deposited to give a black or brown band. Silver stains may commence immediately after electrophoresis, or, alternatively, after staining with CBB. With the latter approach, the major bands on the gel can be identified with CBB and then minor bands, not detected with CBB, resolved using the silver stain. The silver stain is at least 100 times more sensitive than CBB, detecting proteins down to 1 ng amounts. Other stains with similar sensitivity include the fluorescent stains SYPRO ® Orange (30 ng) and SYPRO ® Ruby (10 ng). These dyes have the advantage of binding to proteins in a reversible manner; a useful property for downstream analysis by, for example, mass spectroscopy.

Glycoproteins have traditionally been detected on protein gels by use of the peri odic acid–Schiff (PAS) stain. This allows components of a mixture of glycoproteins to be distinguished. However, the PAS stain is not very sensitive and often gives very weak, red-pink bands, difficult to observe on a gel. A far more sensitive method used nowadays is to blot the gel and use lectins to detect the glycoproteins. Lectins are protein molecules that bind carbohydrates, and different lectins have been found that have different specificities for different types of carbohydrate. For example, certain lectins recognise mannose, fucose or terminal glucosamine of the carbohydrate side-chains of glycoproteins. The sample to be analysed is run on a number of tracks of an SDS–polyacrylamide gel. Coloured bands appear at the point where the lectins bind if each blotted track is incubated with a different lectin, washed, incubated with a horseradish-peroxidase-linked antibody to the lectin, and then peroxidase substrate added. In this way, by testing a protein sample against a series of lectins, it is possible to determine not only that a protein is a glycoprotein, but to obtain information about the type of glycosylation.

Quantitative analysis (i.e. measurements of the relative amounts of different proteins in a sample) can be achieved by scanning densitometry. A number of commercial scanning densitometers are available, and work by passing the stained gel track over a beam of light and measuring the transmitted light; standard office desktop scanners can also be used for this purpose. A graphics presentation of protein zones (peaks of absorbance) against migration distance is produced, and peak areas can be calculated to obtain quantitative data (e.g. using the software ImageJ). However, such data must be interpreted with caution because there is only a limited range of protein concentrations over which there is a linear relationship between absorbance and concentration. Also, equal amounts of different proteins do not always stain equally with a given stain, so any data comparing the relative amounts of protein can only be semi-quantitative. An alternative way of obtaining such data is to cut out the stained bands of interest, elute the dye by shaking overnight in a known volume of 50% pyridine, and then to measure spectrophotometrically the amount of colour released. More recently gel documentation systems have been developed, which are replacing scanning densitometers. Such benchtop systems comprise a video imaging unit (computer linked) attached to a small ‘darkroom’ unit that is fitted with a choice of white or ultra violet light (transilluminator). Gel images can be stored on the computer, enhanced accordingly and printed as required on a printer, thus eliminating the need for wet developing in a purpose-built darkroom, as is the case for traditional photography.

Although gel electrophoresis is used generally as an analytical tool, it can be utilised to separate proteins in a gel to achieve protein purification. Protein bands can be cut out of protein blots and sequence data obtained by subjecting the blot to mass spectrometric analysis. Stained protein bands can be cut out of protein gels and the protein recovered by electrophoresis of the protein out of the gel piece (electroelution). A number of different designs of electroelution cells are commercially available, but perhaps the easiest method is to seal the gel piece in buffer in a dialysis sac and place the sac in buffer between two electrodes. Protein will migrate out of the gel piece towards the appropriate electrode, but will be retained by the dialysis sac. After electroelution, the current is reversed for a few seconds to drive off any protein that has adsorbed to the wall of the dialysis sac and then the protein solution within the sac is recovered.

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مواضيع ذات صلة

Protein (Western) Blotting

Cellulose Acetate Electrophoresis

Other Methods of Protein Purification

Chromatographic Methods for Protein Purification

Producing Recombinant Protein

Engineering Proteins for Purification

حوامض أمينية محورة Modified Amino Acids

Amplification methods

Immunoelectrophoresis

Immunoprecipitation

Antibody production

Introduction to Immunological methods