Transfer buffer 1 can be stored at room temperature for several weeks. The pH of the buffer should be ~9.2 and does not need any adjustment. Use Coomassie dye only in gels after transfer to check the efficiency of the transfer, or if you have no plans to transfer it and just want to observe the results of the separation between SDS-PAGE. When no loading or transfer has even occurred, load control bands can be used to quantify the amounts of protein in each lane.
The balance of SDS and methanol in the transfer buffer, the size of the protein and the percentage of gel may affect transfer efficiency. Detailed instructions for the transfer process are found on the websites of the manufacturers of the transfer devices, and will vary depending on the system. If the membrane in the ice-cold transfer buffer is not balanced, a contraction will occur during the transfer and a distorted transfer pattern will occur. Electrotransfer is almost exclusively the contemporary transfer method of choice because of its speed, transfer uniformity and transfer efficiency.
Electrotransference is based on the same principles of electromobility that drive protein migration during separation in PAGE. In general, wet transfer requires cooling the unit and internal recirculation of the transfer buffer through the presence of an agitation magnet. The proteins will slowly elute from the gel at this point, so do not store the gel and proceed immediately to transfer it. The ratio of Tris and glycine in the transfer buffer is not necessarily the same as for wet transfer; see the device manufacturer's protocol.
The membrane is placed in direct contact with the gel and several layers of filter paper soaked in transfer buffer are placed above and below the gel and membrane. A standard wet transfer buffer is the same as the 1x tris-glycine buffer used as a buffer to operate the gel, but without SDS and with the addition of methanol to a final concentration of 20%. In this transfer choice, both the gel and the membrane are completely submerged in the transfer buffer and a current is applied in the direction of the gel to the membrane. Transfer the gel (keep the dye mixture; it can be reused many times) to a mixture of 67.5% distilled water, 7.5% acetic acid and 25% methanol, place it on a shaker and replace it with a new rinse mixture until excess dye has been removed.
The reduction of methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to be transferred more easily. Just as proteins with an electrical charge (provided by the SDS attached to them) can be induced to travel through a gel in an electric field, proteins can also be transferred in an electric field from the gel to a robust support, a membrane that removes proteins from the gel.