Protein Gel and Membrane Stains

Coomassie dyes (also referred to as Coomassie blue or Coomassie brilliant blue) are common dyes used for the visualization of proteins separated by protein gel electrophoresis. Coomassie based stains offer easy staining protocols and provides good quantitative linearity with good sensitivity. Coomassie based dyes do not permanently chemically modify the target proteins rather interact via non-covalent interactions. Because no chemical modification occurs, excised protein bands can be completely de-stained and the recovered proteins can be used for analysis by mass spectrometry or sequencing.

In acidic conditions, Coomassie binds to basic and hydrophobic residues of proteins, changing in color from a dull reddish-brown to intense blue. As with all staining methods, Coomassie staining detects some proteins better than others, based on the chemistry of action and differences in protein composition. Thus, Coomassie staining can detect as little as 8–10 ng per band for some proteins and 25 ng per band for most proteins.

Two forms of the dye exists- R-250 (R standing for the reddish tint) and G-250 (G standing for the greenish tint). Both variants offer the same relative sensitivity and easy detection. Coomassie G-250 (also known as colloidal Coomassie dye) offers a faster staining protocols and eliminates the need for de-staining.

Silver staining is the most sensitive colorimetric method for detecting total protein. The technique involves the deposition of metallic silver onto the surface of a gel at the locations of protein bands. Silver ions (from silver nitrate in the staining reagent) interact and bind with certain protein functional groups. The strongest interactions occur with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines (Lys). Various sensitizer and enhancer reagents are essential for controlling the specificity and efficiency of silver ion binding to proteins and effective conversion (development) of the bound silver to metallic silver. The development process is essentially the same as for photographic film: silver ions are reduced to metallic silver, resulting in a brown-black color.

Silver staining protocols require several steps, which are affected by reagent quality as well as incubation times and thickness of the gel. An advantage of commercially available silver staining kits is that the formulations and protocols are optimized and consistently manufactured, helping to maximize consistency of results from experiment to experiment. Kits with optimized protocols are robust and easy to use, detecting less than 0.5 ng of protein in typical gels.

Silver stains use either glutaraldehyde or formaldehyde as the enhancer. These reagents can cause chemical crosslinking of the proteins in the gel matrix, limiting compatibility with de-staining and elution methods for analysis by mass spectrometry (MS). Therefore, optimization of sensitivity vs. protein recoverability is critical when employing silver staining as part of an MS workflow.

Silver stain formulations can be made such that protein bands stain black, blue-brown, red, or yellow, depending on their charge and other characteristics. This is particularly useful for differentiating overlapping spots on 2D gels.

Fluorescent stains offer high sensitivity combined with simple staining protocols. Majority of fluorescent stains offer sensitivities similar to that obtained with silver staining techniques and can be used as a good alternative to silver staining in 1D or 2D electrophoresis. Unlike silver stains, fluorescent stains require equipment for visualization. Typical stains can be viewed with a standard UV or blue/green-light trans illuminator or with imaging equipment equipped with the appropriate filters. Fluorescent protein gel stains offer linear quantitation ranges, with some stains reporting over three orders of magnitude.