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.
Imaging and Quantitation
Processes such as quality control of therapeutic proteins and monitoring of protein expression rely on reproducible quantitation of protein PAGE bands, which in turn relies on the quality of the method used to visualize the protein. Variables in Coomassie staining/destaining conditions such as agitation times, volumes, solution changes, and temperature affect the reproducibility of results. Uneven staining of the gels may result in erroneous quantitation of the protein bands. In contrast, using the Criterion Stain Free Imaging System, gels have a uniform and low background level and yield consistent and reproducible results.
To compare the reproducibility of quantitation using Coomassie staining versus the Criterion Stain Free Imaging System, serial dilutions of a PAGE protein standard were run on polyacrylamide gels in quadruplicate, visualized with UV irradiation, and subsequently stained with Coomassie G-250.
The quantity of protein present in the b-galactosidase band (MW 116,000) was estimated using Bio-Rad’s Image Lab automated image-analysis software. Coomassie staining caused a higher variability of quantitation, which rendered a coefficient of variation (CV) of 19.7%, versus a CV of only 7.8% using the Criterion Stain Free Imaging System.
Extending this analysis across all of the bands in the protein standard, the Criterion Stain Free Imaging System demonstrated superior or comparable limits of detection (LODs; 0.2 to 5 ng) and limits of quantitation (LOQs; 0.5 to 6 ng) for all of the proteins except aprotonin, which does not contain tryptophan (Figure 1).
In most organisms, proteins without tryptophan represent less than 10% of the proteins from 10–260 kD, the separable range for most PAGE gels. Examination of predicted proteomes for common experimental organisms shows that proteins lacking tryptophan are biased toward small molecular weight. In fact, the percentage of human proteins larger than 10 kD that lack tryptophan is only 7.3%. The Criterion Stain Free Imaging System can be used with a variety of complex protein samples to form a number of sources, with the results being visually equivalent to Coomassie staining.