Differential staining methods for specific protein modifications such as glycoproteins and phosphoproteins are available. Sometimes it is desirable to detect a subset of proteins rather than all of the proteins in a sample. This is particularly useful for differentiating overlapping spots on 2D gels. Silver stain formulations can be made such that protein bands stain black, blue-brown, red, or yellow, depending on their charge and other characteristics. protein recoverability is critical when employing silver staining as part of an MS workflow. Therefore, optimization of sensitivity vs. These reagents can cause chemical crosslinking of the proteins in the gel matrix, limiting compatibility with destaining and elution methods for analysis by mass spectrometry (MS). Silver stains use either glutaraldehyde or formaldehyde as the enhancer. Kits with optimized protocols are robust and easy to use, detecting less than 0.5 ng of protein in typical gels. 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. Silver staining protocols require several steps, which are affected by reagent quality as well as incubation times and thickness of the gel. Because no chemical modification occurs, excised protein bands can be completely destained and the proteins recovered for analysis by mass spectrometry or sequencing. Then, the staining reagent is added, usually for about 1 hour finally, a water or simple methanol: acetic acid destaining step is used to wash away excess unbound dye from the gel matrix. ![]() ![]() An initial water wash step is necessary to remove residual SDS, which interferes with dye binding. Thus, Coomassie staining can detect as little as 8–10 ng per band for some proteins and 25 ng per band for most proteins.Ĭoomassie dye staining is especially convenient because it involves a single ready-to-use reagent and does not permanently chemically modify the target proteins. As with all staining methods, Coomassie staining detects some proteins better than others, based on the chemistry of action and differences in protein composition. In acidic conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing in color from a dull reddish-brown to intense blue.
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