Post Electrophoretic Analysis
Silver Staining Protein Gels
Utilizing the same chemistry as black and white photography, silver staining is another highly sensitive method for the visualization of protein bands on electrophoresis gels. Silver ions are reduced to insoluble silver metal granules in the vicinity of the protein molecules. Sufficient silver deposition is visible as a dark brown or black band on the gel.
The exact mechanism of silver staining is subject to debate, but certain key points are generally acknowledged. Staining appears to be dependent upon the initial reduction and resulting immobilization of a small number of silver ions by the proteins in the gel. This reduction may be caused by aldehydes created during fixing with glutaraldehyde or with oxidizing agents (e.g. chromic acid). The reduction of silver may also be enhanced by sequestration of the silver cations by carboxylic acid side chains, or by amino side chains, creating an amine-silver complex.
The initial deposition of silver is referred to as a latent image. This "image" is not visible, as the number and size of the silver granules deposited at this stage are minute. In the next stage, a moderate reducing environment is created in the gel, in which all of the silver ions begin to “plate out” slowly. The silver granules which make up the latent image act to catalyze this process, resulting in more rapid deposition of silver at the sites of the protein bands. Silver staining is thus a kinetic process: development must be monitored and stopped at the point where the highest contrast between band and background is achieved.
Silver Staining with the Sterling Silver Kit
For mini-gels (10 X 7 cm), use 100ml of each solution. For larger gels, increase STERLINGvolumes appropriately to immerse gel to a depth of 1cm. Wash mini-gels in 200ml volumes of water, and agitate continuously during all steps. Glassware must be clean, and the water should be distilled or high-quality deionized.
- Incubate the gel for 25 minutes in 100ml of the standard mixture of 5:5:1 methanol:water: acetic acid.
- Decant fixative, then add reconstituted STERLING Fixative (45ml water, 50ml methanol, 5ml STERLING Fixative Concentrate) and fix for an additional 5 minutes.
- Rinse the gel twice for 15 minutes in deionized water. The addition of 0.1% nonionic surfactant will aid in submerging the gel.
- While the gel is washing, prepare a staining solution (see below). Do not combine the two-component solutions until just prior to use.
STAINING SOLUTION PREPARATION
- Dilute 25ml Reagent A with 25ml of water.
- Dissolve 2.8 grams of Reagent B in 50ml of water. STIR UNTIL COMPLETELY DISSOLVED (approx. 5-10 minutes).
- Immediately prior to use, quickly pour the mixture from Step 1 into the mixture from Step 2 while stirring, and pour over gel. The combined solution has a useful life of 20 minutes.
- Decant wash solution and immerse gel in combined staining solution.
- Bands will begin to appear in 5-10 minutes. When desired intensity is achieved, stop development by immersing the gel in a 5% acetic acid solution.
NEXT TOPIC: Guide Strip Technique
- UV Shadowing
- Uneven Staining
- Staining Proteins Immobilized on Membranes
- Staining Protein Gels with Coomassie Blue
- Southern Blotting
- Smeared Bands
- Silver Staining Protein Gels
- Silver Staining DNA Gels
- Protein Fixation on Gels
- Post-Electrophoretic Visualization with Nuclistain
- Overview of Western Blotting
- Northern Blotting
- Method for Western Blotting
- Mechanism of Immunostaining
- Mechanism of Immunostaining
- Immunostaining with Alkaline Phosphatase
- Guide Strip Technique
- Faint bands, low background
- Faint Bands, High Background
- Ethidium Bromide Staining
- Enzyme Linked Immunosorbent Assay (ELISA)
- Coomassie Blue Stain- Troubleshooting
- Blotches on Gel
- Autoradiographic Enhancement with Autofluor
- An Overview of Northern and Southern Blotting
- Alkaline Blotting
- Sample Preparation
- Electrophoresis Buffers
- Blotting and Detection Buffers
- Gel Matrices
- Gel Accessories
- Ultra Pure Reagents