Nitrocellulose is prewetted by floating and then immersing in dH20 or transfer buffer, and PVDF is prewetted in methanol. Prior to assembly in the transfer apparatus, the blotting membrane is prewetted. Usually a corner is marked for later orientation, though this is not necessary when using pre-stained molecular weight markers in one of the side lanes.

Since oils present on the hands will interfere with signal on the blotting membrane, it is advisable to wear gloves while handling the blotting membrane. Often the blotting membrane is supplied precut to size, or else it is precisely cut to match the size of the gel. While the gel equilibrates in transfer buffer, the blotting membrane is prepared. Sometimes higher background staining is seen with PVDF membranes, and extra care must be taken to prevent this from occurring.įigure 8: Western Transfer Methods Setting Up the Transfer PVDF demonstrates superior mechanical strength making it suitable for stripping/reprobing and for further protein characterization techniques, such as sequencing and proteolysis. However, nitrocellulose is brittle and thus it is usually less effective when blots need to be reused. Nitrocellulose has been in use for a long time, and is sometimes preferred because of its excellent protein binding and retention capabilities. The solid support onto which the separated proteins are transferred is usually of two types, nitrocellulose or polyvinylidene fluoride (PVDF) membrane, both of which bind proteins with high affinity. Since there will be significant variation in the chosen transfer system, it is best to consult the manufacturer of the equipment used for specific instructions. Wet transfer is usually considered to be more reliable because it is less likely to dry out the gel, and is often preferred for larger proteins.

Transfer times vary from 1 hour (semi-dry transfer) to several hours or overnight (wet transfer). With semi-dry transfer, the gel/blotting paper/filter paper sandwich is assembled on large electrode plates which generate the electric field, and buffer is confined to the stack of wet filter papers. The cassette is then immersed in a buffer tank and subjected to an electrical field. In a wet transfer, the gel/blotting paper/filter paper sandwich is placed into a cassette along with protective fiber pads.

Electrophoretic transfer can be accomplished under wet or semi-dry conditions. The transfer buffer used for electroblotting is similar to gel running buffer with the addition of methanol which helps proteins bind to the blot. It is imperative that the membrane is placed between the gel and the positive electrode so that the negatively charged proteins migrate from the gel onto the membrane. The gel and blotting membrane are assembled into a sandwich along with several sheets of filter paper which protect the gel and blotting membrane and help to ensure close contact between their surfaces. The electric field used for the transfer is oriented perpendicular to the surface of the gel causing proteins to move out of the gel and onto the blotting membrane, which sits between the gel surface and the positive electrode. While it is possible to use diffusion or vacuum assisted transfer, electroblotting (Towbin et al.,1979) is the method relied upon in most laboratories, due to the speed and efficiency of transfer. Following gel electrophoresis, the separated protein mixtures are transferred to a solid support for further analysis.

Bio rad semi dry transfer