Western Blotting is used for the detection of a specific protein using immunodetection. The process involves the immobilization of a protein of interest to a membrane, PDVF or Nitrocellulose. Once the protein is bound to the membrane it is then probed with a primary antibody and a secondary antibody conjugated to an enzyme (HRP or AP). The Blocking step is crucial in that it prevents nonspecific binding interaction on the membrane. Blocking is achieved by placing the membrane in a blocking solution; the blocking solution which consist of proteins, blocks the charges that would attract the antibodies. There are a variety of blocking agents used in this process they include BSA, dried milk powder, non-animal protein , and protein free blocking reagents. The latter two would theoretically reduce the amount of cross-reactivity of animal proteins. Choosing the correct blocking reagent based on the protein being targeted is important. For instance, trying to detect a phosphoprotein with anti-phosphotyrosine antibodies might require a protein free blocking agent or a non-animal protein blocking agent; it has been observed that using non-fat milk there is a high level of cross reactivity with phosphoprotein antibodies. Also, BSA can be problematic causing high cross reactivity with phosphor-tyrosine antibodies and biotin. Taking the time to fine tune the blocking step will lead to better sensitivity, and automating the blot processing steps by using the BlotCycler touch or Mini will ensure reduced background and blot to blot reproducibility.
