My research requires the identification of proteins associated with a tagged-protein of interest (HSF) using immunoprecipitation. Initially, I used Coomassie Blue for staining gels, but found that the stained bands were too weak to work with easily. Therefore, I decided to try the Bio-Rad Silver Stain Kit and SYPRO Ruby Protein Stain, based on increased sensitivity claims by Bio-Rad when compared to Coomassie staining. The kit includes stock oxidizer, stock silver reagent and developer. Other reagents required for the procedure (that are not included) are methanol and acetic acid. Amazingly, this kit contains enough reagent to stain about 25 full size gels or 50 mini gels. Conveniently, individual reagents can be ordered separately. The oxidizer and the silver reagent simply need to be diluted with water and maintained at room temperature, while the developer is weighed out and re-suspended in water (good for 1 month after re-suspension). If the reagents are too cold, it could affect the staining, slowing down the rate of development. I recommend filtering all solutions and using gloves at all times during preparation of the solutions, as well as, staining. Due to the sensitivity of this kit, contamination (i.e. from skin keratin) may cause the appearance of many bands across the entire gel.
With this kit, silver staining of polyacrylamide gels is really quick and easy. It can be done in a couple of hours: 30 min in fixative (40% methanol and 10% acetic acid), 5 min in oxidizer, 15 min of extensive washing with water, 20 min in silver reagent, variable amount of time (5-15 min) in developer and finally, a soak in stop solution (5% acetic acid). If time is limited, the gel can be left in the fixative solution overnight without affecting overall results. The stained bands on the gel are usually dark brown against a pale background and can usually be seen in a matter of minutes. It is necessary to monitor the development of the staining closely because the intensity might increase quickly, resulting in a background that turns yellowish-brown. In addition, this kit can be used to stain nucleic acids (PAGE gels only), but I have not used it for this purpose. It is supposed to be more sensitive (up to 3 fold) than ethidium bromide.
I am using this kit to try to identify proteins that are associated with HSF in C. elegans since it is not well known in this model system. I am pulling down these proteins by using an antibody covalently attached to affinity beads. Initially, I had a problem with non-specific binding but I have been able to alleviate that problem with changes in the washing protocol. Using this kit, I get about 4 or 5 bands which are specific to the HSF pull down. A couple of them are strong (one of them, based on its size, should be HSF) while the others are relatively weak.
This Silver Stain Kit is not compatible with mass spec analysis. So for now I just want to see if I can pull down and detect proteins using this kit and then use mass spec compatible stain that would allow me to cut out a bands for analysis. I have found this Silver Stain Kit to be many times more sensitive than coomassie staining which has allowed us to identify even very weak bands on the gel.
Overall, I have been very happy with this Silver Stain Kit. Most of the reagents are pre-made and simply require dilution in water. There is a large enough volume of reagents to do many gels. Most importantly, I obtain consistent results very quickly. For the price, this kit is an excellent resource.
Hee Chul Lee
Research Fellow
Internal Medicine
University of Michigan Medical School