The improved SilverSNAP® Stain Kit II is an easy and fast way to detect proteins of low concentration in polyacrylamide gels. In comparison to the former Silver SNAP® Kit, the new kit was improved to reduce time and enhance flexibility: the washing times are reduced and the fixing and staining steps can be extended overnight. To reduce background staining, an additional short incubation in sensitizing solution was included. The SilverSNAP® Stain Kit II contains the sensitizer, enhancer and staining solutions and the developer as stock solutions. Working solutions have to be prepared fresh directly before use according to the instructions. In addition, diluted ethanol and ethanol/acetic acid solutions are needed for the fixation and washing steps. All of the reagents have to be of highest quality and the staining containers clean (protein-free) to avoid high background and spotty staining. Staining and washing steps are performed at room temperature, but the kit should be stored at 4°C.
When staining home-made polyacrylamide gels with the kit it is better to use thinner gels (1 cm and less in thickness), since the fixation and staining solutions might not penetrate completely into the gel during the short incubation times, resulting in weaker staining. The protocol is easy to follow, but it is good to know each step in advance since some of the solutions have to be prepared just before using them. Being prepared allows you to follow the recommended incubation times, which are more critical for some steps than others. For instance, the sensitizer incubation and the following washes in dH2O require very short incubation times, but others, like the fixation and enhancer incubations as well as some washes are not as critical. However, the washing times before the developing step should not be extended as excessive washing washes out the staining solution, which results in fainter bands. Even prolonged incubation of the gel in developer does not enhance faint bands. In cases were faint bands are expected, it is the best to follow the times of the staining protocol exactly. Fixation overnight and incubation of the gel in enhancer/staining solution up to 4 hours did not change the staining intensity. Pierce also recommend to restain following extensive washes after the developing step (see troubleshooting).
I mostly over-stained the gels by leaving them in the developer solution too long (once the bands get visible they darken fast). According to my experience, bands continue to darken even during incubation in the stopping solution, therefore, dark bands are easily over-stained. Therefore, I recommend to quickly stop the developing step once bands become visible. Then quickly rinse the gel once with stop solution to remove the developer solution before incubating it for a longer time in the stop solution. (Note: gels can be stored in the stop solution or dH2O). Care must also be taken to wear gloves when preparing and loading the gel as well as when running and staining the gels in order to avoid contamination with keratin (50-65 kD): the major contaminant from skin and hair.
The SilverSNAP® Stain Kit II is a highly sensitive, fast and easy way to stain proteins in polyacrylamide gels. When unknown protein homogenates are loaded, it is crucial to follow the protocol precisely to avoid results like too faint or over-stained bands. There is a little information about the staining kit and troubleshooting tips on Pierce’s website and in the instruction manual, but the Pierce technical service is an excellent source for tips and technical information.