18 May 2011

Protein Gel Stains

Once protein bands contain been separated by polyacrylamide gel electrophoresis, they can be blotted (transferred) to crust used for analysis by Western blotting (see linked article) or they can be visualized absolutely in the gel using various streak or detection methods.

Coomassie dye is the as a rule widespread reagent used for streak protein bands in electrophoretic gels. In vogue tart safeguard conditions, coomassie dye binds to basic and hydrophobic residues of proteins, changing from dull reddish-brown to intense blue (see before images on this page). At the same time as with all streak methods, Coomassie dye reagents detect various proteins better than others based on their chemistry of war and differences in protein work of art. For as a rule proteins, however, Coomassie dye reagents detect as the minority as 10 nanograms for each crowd in a mini-gel. Thermo Scientific GelCode Blue and Imperial Stains work Coomassie G-250 and R-250 dyes, correspondingly.

Most streak methods implicate various version of the same incubation steps:

1.     A water-wash to remove electrophoresis buffers from the gel matrix
2.     An acid- or alcohol-wash to condition or settle the gel to limit diffusion of protein bands from the matrix
3.     Treatment with the stigma reagent to allow the dye or element to circulated into the gel and truss (or react with) the proteins
4.     Destaining to remove overkill dye from the background gel matrix

Another widespread method used for detecting protein bands surrounded by a gel is silver streak, which deposits clanging silver on top of the superficial of a gel by the location of protein bands. Commercial silver stigma kits are exceptionally robust and painless to work, detecting not as much of than 0.5 nanograms of protein in classic gels. Silver stains work glutaraldehyde or formaldehyde as enhancers, and classic formulations chemically crosslink proteins in the gel matrix. This limits the efficiency of destaining and recovery of proteins used for downstream applications, such as collection spectrometry (MS). Thermo Scientific Silver Stain is fully compatible with destaining and elution methods obligatory used for MS analysis.

Another streak method compatible with protein recovery and collection spec analysis is the Pierce Zinc Reversible Stain. The Zinc stigma is unique in so as to it does not stigma the protein absolutely, but as a replacement for results in an obscure background with release, unstained protein bands. The bands can be photographed by introduction a dark background behind the gel. Zinc streak is as precision as classic silver streak (detects < 1 ng of protein) and is definitely erased, allowing trouble-free downstream analysis by collection spectrometry or Western blotting.

In vogue current years, the demand used for fluorescent stains has increased with the improvements and popularity of fluorescence imaging equipment. Fluorescent stains are at the present accessible with excitation and giving out maxima corresponding to the for all filter sets and laser settings of as a rule fluorescence imagers. Thermo Scientific Krypton Stains are state-of-the-art fluorescent protein stains.

Finally, several traditional and innovative chemistries exist used for streak feature classes of proteins in polyacrylamide gels. These include stigma kits to detect glycoproteins or phosphoproteins.

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