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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