Silver staining is a method for visualizing proteins in polyacrylamide gels, with the important advantage that it is up to 100 times more sensitive than staining with Coomassie blue. GE Healthcare’s PlusOne® Silver Staining Kit, Protein can be used to stain conventional or precast gels, either denaturing or nondenaturing, and isoelectric focusing gels. The kit provides concentrated solutions and packets of powdered reagents that are brought up to the desired volume using distilled water. Some solutions require ethanol, acetic acid, or glycerol, which are not provided in the kit.
The polyacrylamide gel is fixed after electrophoresis, followed by steps for sensitizing, incorporating silver, color developing, and stopping, with some additional wash steps in distilled water. All steps are conducted at room temperature. Some of the solutions (such as the stop solution, which contains EDTA) require a few minutes of stirring on a magnetic plate. The protocol takes about 3 hours including a 1-hour preserving step that prepares the gel to be dried overnight in cellophane (not provided). According to the product literature, the PlusOne® Silver Staining Kit, Protein is also compatible with the Hoefer® Automated Gel Stainer, and a separate protocol is provided for use with Ampholine PAGplate precast gels, which come with a plastic backing.
The PlusOne Silver Staining Kit, Protein contains materials for 10 large gels (12 x 26 cm) or 20 mini-gels (10 x 10 cm). However, the packets containing premeasured powdered reagents such as EDTA are designed for use with large gels (12.5 x 26 cm). Using the kit for 10 x 10 cm mini-gels necessitates either wasting reagents or measuring out part of the packet. The solutions can be made up to 24 hours in advance, but some components (for example formaldehyde) must be added immediately before use. The kit has an additional advantage that I have discovered from experience: I have incubated gels overnight in the sensitizing solution with no loss of sensitivity or other bad effects, which is very convenient after spending the day preparing samples and running the gel.
I have used GE Healthcare’s PlusOne® Silver Staining Kit, Protein many times with reliably excellent results. The silver stained bands range from amber to dark brown in color depending on the amount of protein present, and are very sharp and distinct. Background is practically nonexistent unless you overdevelop the gel. The kit is excellent for detecting small amounts of protein; however, because the method is so sensitive, overloading of protein can make it difficult to detect bands of interest. The silver staining method has one drawback compared to Coomassie blue staining: waste disposal is more complicated. Three of the solutions contain toxic materials (for example, glutardialdehyde) that may require disposal as hazardous waste.
Kimi Nishikawa
Graduate Student
State University of New York at Albany