Protein gel staining methods: an introduction and overview.
Laboratory scientists who encounter protein biochemistry in many of its myriad forms must often ask: is my protein pure? The most frequent response: run a denaturing SDS polyacrylamide gel. Running this gel raises another series of considerations regarding detection, quantitation, and characterization and so the next questions invariably center on suitable protein gel staining and detection methods. A total protein profile can be determined with the colorimetric methods embodied in Coomassie Blue and silver staining methods, or increasingly, with fluorescent stains. Protein quantitation can be done following staining, with fluorescence- and instrumentation-based methods offering the greatest sensitivity and linear dynamic range. Protein posttranslational modifications such as phosphorylation and glycosylation can be reliably determined with several fluorescence-based protocols. Staining and detection with two or more different stains can be done in series to establish relative profiles of modified versus total protein or to assess purity at two levels of quantitative sensitivity. The choice of staining method and protocol depends on the required rigor of detection and quantitation combined with available instrumentation and documentation capabilities. Other considerations for staining methods include intended downstream analytical procedures such as mass spectrometry or peptide sequencing, which preclude some methods. Nonfixative staining methods allow western blotting after gel staining. Laboratory custom and budget or intellectual curiosity may be the ultimate determinate of the chosen gel staining protocol.
Steinberg
Thomas H
TH
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*
D000180:Acrylic Resins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D010750:Phosphoproteins; D011506:Proteins; C016680:polyacrylamide gels