For proteins larger than 100 kDa, it is recommended that SDS is included at a final concentration of 0.1%. The negatively-charged proteins travel towards the positively-charged electrode, but are bound by the membrane, preventing them from continuing on.Ī standard buffer for wet transfer is the same as the 1x Tris-glycine buffer used as the gel running buffer, but without SDS and with the addition of methanol to a final concentration of 20%. The sandwich is submerged in transfer buffer to which an electrical field is applied. In a wet transfer, the gel and membrane are sandwiched between sponge and paper (sponge > paper > gel > membrane > paper > sponge) and all are clamped tightly together to ensure that no air bubbles form between the gel and membrane. The two are sandwiched between absorbent materials, and the sandwich is clamped between solid supports to maintain tight contact between the gel and membrane. For both kinds of transfer, the membrane is placed next to the gel. Wet transfer is less prone to failure due to drying of the membrane, and is especially recommended for large proteins. Transfer can be done using a wet or semi-dry system. Early methods relied on diffusion blotting in an electrical field is now standard. Just as proteins with an electrical charge (provided by the SDS bound to them) can be induced to travel through a gel in an electrical field, so can the proteins be transferred in an electrical field from the gel onto a sturdy support, a membrane that "blots" the proteins from the gel. 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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