My research is focused on the identification of proteins associated with HSF in C. elegans. Most of my experiments involve immunoprecipitating a tagged version of HSF to identify associated proteins. As a way to initially identify these proteins, I run the immunoprecipitation reactions out on an SDS PAGE gel and then stain the gel. For staining the gel, I have been using the SYPRO Ruby Protein Gel Stain from Bio-Rad. The SYPRO Ruby Gel Stain is a fluorescent stain for detecting proteins separated by acrylamide gel electrophoresis (1-D or 2-D). The staining dye is comprised of an organic component and a heavy metal and is stable for 6 months at room temperature. It comes as a 1x pre-mixed solution in three sizes, 5 L, 1 L and 200 ml. The 200 ml volume (which we ordered) can stain about 4 mini gels (50 ul of sample/gel) according to their manual, but I have been using 30ul of sample/gel and still get very good staining. Additional items required, but not provided, are methanol, acetic acid and a UV transilluminator.
Staining with SYPRO is quick and the instruction manual outlines a simple protocol for staining polyacrylamide gels of different sizes. Basically, after running the gel, it is fixed in 10% methanol and 7% acetic acid. The mini-gel is then covered with about 30ml of the SYPRO Ruby protein gel stain for about 3 h (alternatively, at this point, the gel can be soaked overnight). After fixation, the gel is washed with 10% methanol and 7% acetic acid for about an hour (replacing the wash solution every 15 minutes or so), followed by rinsing with deionized water. Finally, the stained protein bands on the gel are visualized using a UV transilluminator. The SYPRO Ruby protein gel stain has two excitation peaks at 280 and 450 nm and has emission maxima near 610 nm. Because SYPRO has good photostability and a long emission lifetime, it can be exposed for a long period of time while still minimizing background fluorescence.
In my experiments, where I am pulling down proteins using an HSF-1 antibody covalently attached to Bio-Rad Affi-Gel beads, the SYPRO stain works very well. Initially, analyzing these samples using a silver stain procedure, multiple bands that appeared to be HSF-specific were revealed. Unfortunately, even though silver staining is very sensitive, it is not mass spec compatible. Since I know now that I can pull down HSF-specific proteins in my system, I am working with the SYPRO Ruby protein gel stain, which is mass spec compatible (allowing the bands to be cut out of the gel to be sent for analysis). The sensitivity of SYPRO Ruby is not as good as Silver Staining. I still see the two bands which were strong on the silver stained gel, but they are not as strong in intensity. The other weak bands I observed on the silver stained gel are not present with the ruby protein staining. So, basically right now I am trying to work out the pull down conditions so that, hopefully, I can observe those weak bands using this staining kit.
Staining with SYPRO Ruby is simple and relatively quick, but does take longer than silver staining (similar to the amount of time it takes to stain with Coomassie Blue). The sensitivity of SYPRO is slightly better than Coomassie, but less than silver stain. Unlike silver staining, it is difficult to see the weak protein bands but I also do not have the background problems that I observe with silver stain. Also, SYPRO Ruby does not interfere with analysis of proteins by Edman-based sequencing or mass spectrometry. Due to the compatibility, I recommend this product if one needs to do analysis by mass spectrometry.
Hee Chul Lee, Ph.D.
University of Michigan Medical School