At Rockland, we routinely perform western blots for analysis of gene expression, antigens, and antibodies. Positive reactivity can be evidenced by the signal generated from a reporter enzyme or fluorophore conjugated to a secondary antibody that recognizes the primary antibody. WB requires successive steps including transfer of the PAGE-separated proteins onto the membrane using either a wet or semi-dry system, pre-incubation on a blocking buffer that will help to reduce non-specific background signal, and incubation with a primary antibody that specifically binds the antigen of interest. Furthermore, alcohols or other reagents can alter or modify molecules andmay therefore destroy some antigenic determinants.Western blot (WB) or immunoblotting is a rapid and sensitive technique that uses antibodies for the specific detection of proteins separated by polyacrylamide gel electrophoresis (PAGE) and immobilized onto nitrocellulose, nylon, or PVDF membrane. Because SDS is not rapidly removed from the protein in the absence of alcohol, the detergent-bound proteins are all initially negatively charged and a more quantitative transfer of proteins is achieved. However, 10 mM 3-1-propanesulfonic acid (pH 9.0 or 11.0) plus 10 % methanol is suggested by Matsudaira for transfers for SDS-PAGE mini-gels to PVDF membrane.Īlthough many variations of electrophoretic transfer of protein to nitrocellulose have been described, we have found that the procedure that omits alcohol from transfer solutions is generally optimal. The recirculating, ice-cooled, high ionic strength buffer used helps prevent the gel from swelling in the absence of methanol during transfer, which can cause poor resolution of proteins on the membrane. The procedure of Towbin as modified by Anderson specifies a Tris-glycine pH 8.3 buffer containing SDS. The choice of buffer composition depends on the types of gel and membrane selected. I looked up the section on protein blotting and immunodetection in "Methods in Enzymology" Vol 182 pp 681-682. We make our world significant by the courage of our questions and by the depth of our answers. For more open-ended questions, try /r/AskScienceDiscussion | Sign up to be a panelist!. ![]() ![]()
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