The principle is the same in each case though. Move the gel to a dish of transfer buffer before proceeding with transfer according to the transfer apparatus manufacturer's instructions.ĭetailed instructions for the transfer process can be found on the websites of the manufacturers of transfer apparatus, and will vary depending on the system. Gels may be destained completely by repeated washing in 0.1–0.25 M Tris/0.25 M EDTA pH 8.0. Proteins come up as clear zones in a translucent blue background. Wash the gels briefly in de-ionized water, and view them against a dark-field background. However, it remains strongly bound to the proteins in the gel, and these take on a deep blue color.īriefly rinse freshly-electrophoresed gels in distilled water (30 sec maximum) and then transfer to a solution of 0.3 M CuCl 2 for 5–15 min. The stain will not bind to the acrylamide, and will wash out (leaving a clear gel).
Transfer the gel (save the dye mixture it can be re-used many times) to a mixture of 67.5% distilled water, 7.5% acetic acid, and 25% methanol, place on shaker, and replace with fresh rinse mixture until the excess dye has been removed. Incubate for 4 h to overnight at room temperature on a shaker. To visualize the fixed proteins place the gel in the same mixture of water/acetic acid/methanol but with the addition of 0.25% by weight Coomassie Brilliant Blue R-250. To prevent diffusion of proteins treat the gel with a 40% distilled water, 10% acetic acid, and 50% methanol solution which causes almost all proteins to precipitate (become insoluble). Only use the Coomassie stain on gels post-transfer to check the efficiency of the transfer, or if you have no plans to transfer and just want to observe the results of the SDS-PAGE separation.Īs soon as the power is turned off the separated protein bands will begin to diffuse (they are freely soluble in aqueous solution). Use the copper stain if you plan to transfer the separated proteins to a membrane, as the Coomassie stain is not reversible. Protein visualization at this stage is useful to determine if proteins have migrated uniformly and evenly.
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