Many methods are available for the detection of the protein bands in the gel after electrophoresis. As there is a need for methods having high sensitivity, ease to use, staining protein quantity in linear range and which do not influence any further protein analysis like Edman degradation or MS analysis, different techniques and protocols are being explored.
Staining is one of methods to visualize proteins on the gel. When the dye interacts with amino acids, proteins become visible and provide information about the physical properties like molecular weight, protein amount or separation efficiency within the electrophoretic system.
Most common method for staining is using coomassie dye which binds directly to basic and aromatic side chains of proteins giving intense blue colour. Prior to the MS analysis, the Coomassie dye has to be removed and it detects in the range of 25- 50 ng per band of protein.
Another popular method is silver staining which is 10 to 100 fold more sensitive depending on different proteins.
Silver staining was introduced in 1979 by Switzer et al, a technique that is sensitive upto 0.3-10 ng level. It does vary, however, with the glycosylation and physical properties of the proteins. Several classes of highly negative charged proteins, including proteoglucans and mucins, which contain high levels of sulfated sugar residues, and some of very acidic proteins are detected poorly by silver staining.
The principle of silver staining is similar to developing photographs.
The complexing power of the proteins for silver ion is utilized here by using a very weak developer based on formaldehyde at alkaline pH, and by rinsing the gel after the silver impregnation and before development. During the rinse, the silver ion or complex will diffuse out of the gel much faster from the places where the complexation is weak (background) than from the places where the complexation is strong (protein). The key of silver staining lies in the fact that there is a developing point where the resulting excess of silver ion at the protein level will increase the speed of reduction to a point where it will overcome the slowing effect due to complexation. Therefore, the reduction will occur where proteins are present and it will ensure a positive image through the autocatalytic phenomenon.
Here we will try to understand this phenomenon in a better way to know how actually gels can be turned into photogenic marvels!!
Two general classifications are 1) silver amine or alkaline method, and 2) silver nitrate or acidic method. Detection levels of proteins depends on how quickly background develops and silver amine with low background is more sensitive but use of glutaraldehyde in this method is a drawback as it is not compatible matrix-assisted laser desorption/ionization mass spectrometry (MALDI - MS).
Basically staining protocol is divided into five main steps.
Fixation is the first step; the gel is fixed to eliminate the interfering substances otherwise giving a high background and poor contrast e.g. SDS, ampholytes and Tris.
In the second step of sensitization, the gel is treated with an agent which will eventually increase the sensitivity.
For sensitivity, the gel is treated with a reagent which is adsorbed by the proteins and which then binds silver complexes providing extra binding sites for the silvering agent. Upon development, this extra silver ion will be reduced, which will increase the sensitivity. Various sensitizers like sulphides, reducing agents, aldehydes have been used but the contrast and the sensitivity of silver staining remained mostly inadequate because minute silver deposition at the background level gave strong background due to autocatalysis, imposing termination before optimal sensitivity can be reached. Thus the best method found was to use very low amounts of thiosulfate during development which at neutral and basic pH forms very strong complexes with silver, not prone to reduction in the weak conditions used for silver staining. Therefore, just adding micromolar amounts of thiosulfate in the developing solution, there is a dramatic reduction of the background.
In the third step of staining, silvering agent is added. Two main families of silver staining methods can be distinguished at this step, those using silver nitrate solutions as the silvering agent (acidic methods) and those using a basic silver-ammonia or less frequently a silver-amine complex (basic methods).
The image is developed, generally by a dilute formaldehyde solution in the fourth step of development. This step is critical and should be carefully timed. Too much rinse will eventually decrease the silver ion concentration and lead to a reduced sensitivity, whereas insufficient rinsing will give a high background.
In the last step, the reaction is stopped to prevent overdevelopment and the gel is prepared for storage.
Many different protocols for silver staining have been suggested by different groups. Each protocol has different advantages regarding timing, sensitivity, cost, and compatibility with other analytical methods, especially mass-spectrometry (MS). Basic procedure is as follows:
Fixation: The gel is washed in solution of 50 ml Ethanol, 12 ml Acetic Acid, 38 ml dH2O and 50 ul of 37% formaldehyde for an hour on shaking.
Washing: After discarding fixation solution, the gel is washed with 50% ethanol/ methanol for 20 minutes twice.
Sensitization: Treat the gel with 10 mg of sodium thiosulfate (Na2S2O3) in 100 ml distilled water for exactly 1 min. Discard solution and rinse with distilled water 3 times for 20 seconds each.
Staining: The gel is stained with 100 mg of silver nitrate (AgNO3) and 0.056% formaldehyde in 100 ml distilled water for 10-30 minutes. Rinsing is again done with distilled water for 20 seconds 3 times.
Developing: Bands are developed using 3 g sodium carbonate (Na2CO3), 50ul 37% formaldehyde, and 2 ml of sensitivity solution (sodium thiosulfate solution) in 98 ml of distilled water. When optimum intensity is reached, developing is stopped.
Stopping: 10% acetic acid solution is used to stop developing bands. After 10 minutes, the gel is rinsed with distilled water.
Gel preservation: Polyester-backed gels can be preserved for many years by air-drying and later bands can be cut for PCR at anytime.
Some basic precautions should be taken to develop beautiful gels like always wearing gloves during holding gels, washing for more time after fixation to increase sensitivity, keeping silver solution in dark and using it cold, also not pouring it directly on the gel, try to use polypropylene trays for staining, gels should be fully immersed in the solution in each step and should be kept on rotary shaker for proper exposure of gels to the solution.
Thus, following few steps carefully we can develop wonderful gels with unsurpassed sensitivity and then we can surely say 'my gels are definitely photogenic!'.
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