How does Pierce Silver Stain Kit work?
The gel is washed and fixed to remove electrophoresis buffer salts. The silver stain is added, releasing silver ions that interact with the proteins. The developer is then added causing the protein bands to darken as the bound silver reduces. The color development is stopped by lowering the pH with acetic acid.
Which types of polyacrylamide gels can be stained?
The stain performs very well for most polyacrylamide gel types (i.e., suppliers and buffer systems). Both 1D and 2D polyacrylamide gels can be stained with this kit. The default protocol is optimized for gels that are 1.0mm thick; incubation and wash times may require adjustment to achieve optimal results for gels of other thicknesses.
What level of detection sensitivity can be expected using this kit?
A detection level of 0.4 ng per protein band has been achieved. Most proteins are easily detectable at low-nanogram (1-5 ng) amounts.
How critical are the fixing and staining times in the protocol?
The protocol is quite flexible with regard to fixing and staining times. After gels are fixed or at least 30 minutes, they can be stored in water until the next day. The gel-staining time can range from 5 minutes to 24 hours without incurring additional background.
How critical are the incubation times of other steps in the protocol?
The 1 minute sensitization and 2 x 1 minute water wash steps are important for optimum stain performance. Development time (2-3 minutes) is also critical, and stop solution must be added as soon as desired development occurs.
What reagents and factors affect silver staining performance?
SDS, glycine, amines and phosphates interfere with the staining method; this is why initial wash and fixing steps are necessary. Silver staining is also sensitive to pH conditions, chelators (sequester silver), and strong oxidants or reductants. To avoid these contaminants, use ultrapure water and clean equipment (avoid metal utensils), and avoid touching the gel except at the edges. Prepare working solutions of stain, sensitizer and developer immediately before use.
Can the bands be removed and analyzed by mass spectrometry following staining?
Yes. See Tech Tip #50: Process stained polyacrylamide gels for mass spectrometry. Alternatively, use our Pierce Silver Stain for MS (#24600), which is the same silver stain kit but also includes destaining reagents and a protocol optimized for maximum protein recovery.
Can a coomassie-stained gel be secondarily stained with the silver stain?
Yes. First destain to completely remove background of the coomassie-stained gel. If an acid or methanol destaining solution was used, thoroughly wash the gel with ultrapure water, then proceed with the Pierce Silver Stain Kit protocol.
Can nucleic acids be detected with this kit?
Yes. References in the literature and molecular biology protocols indicate that nucleic acids can be detected in polyacrylamide gels with silver stains of this type.
Can gels be dried after staining?
Yes. Thoroughly wash the gel with water to remove the acetic acid and then soak the gel in a solution containing 5% glycerol and 10% methanol (or 10% ethanol) for one hour before drying in a gel-drying apparatus.