June 15, 2010 (Vol. 30, No. 12)
Kevin McDonald, Ph.D.
New Imaging Technique with Broad Utility Provides Protein Assessment Alternative
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.
The utility of this novel visualization technique will be judged largely on its compatibility with techniques like Western blotting and mass spectrometry. In addition to allowing rapid and direct confirmation of appropriate protein patterns, this technique enables efficient transfer of protein bands to membranes and subsequent immunodetection (Figure 2).
The bands detected on the gel by the Criterion Stain Free Imaging System can subsequently be transferred to membranes, while bands stained with Coomassie typically cannot. The Criterion Stain Free Imaging System eliminates the need to run and Coomassie stain a duplicate gel to assess the protein pattern before transfer to the membrane as well as the need for time- and reagent-consuming traditional methods such as Ponceau S staining to assess protein transfer prior to detection in Western blotting.
The Criterion Stain Free Imaging System enables quick and easy monitoring of the efficiency of transfer by imaging the membrane and gel after transfer. It has been demonstrated that UV activation does not interfere with the transfer or immunodetection of two human proteins when using polyclonal antibodies. The limits of detection and signal intensities are the same, with or without UV irradiation of the gels.
Monoclonal antibodies can also be used if they are not specific for an epitope containing tryptophan. Since tryptophan is typically between only 1 and 2% of the amino acid residues in most proteins, most antibodies will not be affected by Trp modification of the target proteins.
The UV-induced modification of tryptophan residues in gel protein bands also does not interfere with subsequent mass spectrometric identification of the proteins. A large-scale study of a total of 56 spots from two sets of UV-activated 2-D gels resulted in identification of 48, using LC-MS/MS analysis, MALDI-TOF-MS analysis, or both.
Forty-six spots gave the same identities as those extracted from nonirradiated gels. These rates of identification are typical of MS analyses of PAGE. A database search with a dynamic modification of the tryptophan residue helped to assure identification of all peptides containing trp, and may assist in the identification of low-abundance proteins.
The disadvantages that accompany Coomassie staining can be cured through the use of a unique protein visualization technique that eliminates two hours of processing time, provides equivalent or superior sensitivity and reproducibility of quantitation, and is suitable for analysis of a wide range of complex protein samples. Additionally, this imaging method improves the ease of use of Western blotting while maintaining sensitivity of detection, and can be used with MS techniques for protein identification.