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Coomassie Staining Has Some Competition

New Imaging Technique with Broad Utility Provides Protein Assessment Alternative

• Kevin McDonald, Ph.D.

(Page 2 of 2)

Downstream Analyses

Figure 2. Gel images of complex protein samples imaged by UV activation or Coomassie G-250 staining: Lane 1, Precision Plus™ Protein unstained standards (Bio-Rad Laboratories); Lane 2, mouse liver extract (12.5 µg); Lane 3, rat liver (12.5 µg); Lane 4, mouse thymus (12.5 µg); Lane 5, rat thymus (12.5 µg); Lane 6, HeLa cell lysates (12.5 µg); Lane 7 E. coli lysate (67.5 µl); Lane 8, ProteoMiner kit-treated human serum (Bio-Rad Laboratories), 5 µL of a 1:8 dilution; Lane 9, untreated human serum, 5 µL of a 1:80 dilution. Samples were run on a 4–20% Criterion Stain Free Tris-HCl gel (Bio-Rad Laboratories) containing the trihalocompound. The gels were activated and imaged on the Criterion Stain Free system (A), then stained with Bio-Safe G-250 stain (B), followed by imaging on a Molecular Imager GS-800 calibrated densitometer. Click Image To Enlarge +

Figure 2. Gel images of complex protein samples imaged by UV activation or Coomassie G-250 staining: Lane 1, Precision Plus™ Protein unstained standards (Bio-Rad Laboratories); Lane 2, mouse liver extract (12.5 µg); Lane 3, rat liver (12.5 µg); Lane 4, mouse thymus (12.5 µg); Lane 5, rat thymus (12.5 µg); Lane 6, HeLa cell lysates (12.5 µg); Lane 7 E. coli lysate (67.5 µl); Lane 8, ProteoMiner kit-treated human serum (Bio-Rad Laboratories), 5 µL of a 1:8 dilution; Lane 9, untreated human serum, 5 µL of a 1:80 dilution. Samples were run on a 4–20% Criterion Stain Free Tris-HCl gel (Bio-Rad Laboratories) containing the trihalocompound. The gels were activated and imaged on the Criterion Stain Free system (A), then stained with Bio-Safe G-250 stain (B), followed by imaging on a Molecular Imager GS-800 calibrated densitometer.

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.

Conclusion

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.