PlusOne™ Silver Staining Kit, Protein from GE Healthcare

Working with proteins, there is always a need for visualization of proteins or protein mixtures that have been separated by 1D or 2D gel electrophoresis. There are different ways of visualizing proteins, depending on downstream applications like native state analysis or mass spectrometry, or depending on the sensitivity one wants to achieve and the possibility of storing gels for archival purposes. Silver staining is a complex process but produces an excellent and long-lasting record. It shows a high sensitivity which allows detection of proteins down to the nanogram level, which is more sensitive than coomassie based detection methods. Nevertheless, mass spectrometry facilities prefer coomassie stained samples for analysis over silver stained ones, due to protein modifications introduced by reagents used for silver staining.

The PlusOne™ Silver Staining Kit, Protein, from GE Healthcare is equipped with reagents sufficient to stain 10 – 60 polyacrylamide gels depending on size and protocol. The kit protocol describes detailed methods (8 different protocols) for staining precast gels as well as thicker, conventional gels used for non-denaturing or denaturing separation, SDS or urea gels and isoelectric focusing gels. The kit provides ready-to-use solutions and pre-weighed chemicals in single packages which minimize preparation time and maximize the ease of use and reliability. All the reagents can be stored at room temperature which I think is very convenient.

On a regular basis, I use this kit for visualizing complex protein mixtures separated by 2D electrophoresis for downstream analysis using specialized detection software packages. Under some circumstances, I also stained 1D gels using the silver staining technique if it was necessary. All the gels used for my experiments are self-cast polyacrylamide gels with different percentages using standard reagents.

I think the major advantages of this kit are the easy to follow protocols and the reliability, since the reagents and solutions are pre-packaged and ready to use. The way this kit is designed reduces day-to-day variability due to mistakes in preparation of staining solutions which is very important for getting valid 2D electrophoresis results. One problem is the production of large amounts of fluid waste with a mixture of silver and aldehydes.

The silver staining process is quite time consuming, nevertheless, the silver staining kit follows a streamlined protocol which only takes about three hours, depending on the length of the developing step. Before starting the staining process you need additional reagents for the preparation of the ready-to-use staining buffers, namely ethanol, glacial acetic acid, and glycerol. In addition, some of the buffers need glutaraldehyde or formaldehyde added immediately before use. In my experience, this is an often-missed step in reagent preparation which for me resulted in less sensitivity. Therefore, I use extra labels on these buffer bottles as a reminder. On the other hand, by omitting the glutaraldehyde and formaldehyde, the method becomes compatible with mass spectrometry analysis, however, at the expense of sensitivity. An important thing about the silver staining procedure in general, but also about this kit, is to stick to incubation times. This is especially important for the developing step since the silver staining technique has the lowest linear dynamic range of the usually used protein stains.

Taken everything together, I think the silver staining kit is a great tool I don’t want to miss in performing protein research.