SDS-PAGE followed by Coomassie staining is a standard, widely used method to visualize proteins. The relative low cost of these dyes and their ready-made solutions, sensitivity in the 5–50 ng range, and time-tested staining and destaining protocols have been keys to their wide acceptance. Images of lanes of blue bands are universally recognized in the life science research community as PAGE protein separations.
Coomassie staining has its drawbacks, however. Gel staining and destaining with Coomassie can take at least two hours. Coomassie is also known to vary widely in its ability to bind proteins, due at least in part, to its affinity for proteins rich in basic amino acids such as arginine, histidine, and lysine. Glycoproteins, which make up more than half of all proteins, stain poorly with Coomassie dye, and there is at least one report in the literature that Coomassie staining may overstate relative protein quantities in gels. In addition, the user-dependent nature of the process can introduce variability, when comparing results generated by different users. Finally, Coomassie staining can generate large quantities of solvents, some of which are hazardous.
Bio-Rad Laboratories’ Criterion Stain Free Imaging System uses a trihalocompound modification of tryptophan providing a desirable alternative to Coomassie staining for many applications. The system uses standard sample preparation, reagents, and electrophoresis protocols, with the trihalocompound incorporated into standard gel formulations.
After electrophoresis, the gel is subjected to UV irradiation for as little as 2.5 minutes, which activates a covalent reaction between the trihalocompound and tryptophan residues on the proteins in the gel. The resultant adduct of tryptophan is fluorescent when excited by the same UV source. The fluorescent signal is then automatically imaged in less than a few seconds by the Criterion Stain Free Imaging System, which produces an image of proteins in the gel.
The entire electrophoretic separation and gel imaging can be completed in about an hour. Another major advantage of the system is that it lends itself well to automation: the gel can be activated, the digital image captured, and the molecular weight and quantity of each protein band can be calculated by an automated system with the push of a button.