<P> To make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The primary method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane . The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel . An older method of transfer involves placing a membrane on top of the gel, and a stack of filter papers on top of that . The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it . In practice this method is not used as it takes too much time; electroblotting is preferred . As a result of either "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein . Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings . </P> <P> This step visualizes the total protein that has been successfully transferred to the membrane . Total protein staining allows the user to check the uniformity of protein transfer and to perform subsequent normalization of the target protein with the actual protein amount per lane . Normalization with the so - called "loading control" has based on immunostaining of housekeeping proteins in the classical procedure, but is heading toward total protein staining recently, due to multiple benefits . At least seven different approaches for total protein staining have been described for western blot normalization: Ponceau S, stain - free techniques, Sypro Ruby, Epicocconone, Coomassie R - 350, Amido Black, and Cy 5 . In order to avoid noise of signal, total protein staining should be performed before blocking of the membrane . Nevertheless, post-antibody stainings have been described as well . </P> <P> Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent the interactions between the membrane and the antibody used for detection of the target protein . Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein--typically 3--5% bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in tris - buffered saline (TBS) or I - Block, with a minute percentage (0.1%) of detergent such as Tween 20 or Triton X-100 . Although non-fat dry milk is preferred due to its availability, an appropriate blocking solution is needed as not all proteins in milk are compatible with all the detection bands . The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached . Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein . This reduces background in the final product of the western blot, leading to clearer results, and eliminates false positives . </P> <P> During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate, this enzyme drives a colorimetric reaction and produces a color . For a variety of reasons, this traditionally takes place in a two - step process, although there are now one - step detection methods available for certain applications . </P>

In western blotting the non-specific sites on the membrane can be blocked by