![]() Because amino acid sequences vary from protein to protein, western blotting analysis can be used to identify and quantify a single protein in a lysate that contains thousands of different proteins. Antibodies bind to highly specific sequences of amino acids, known as epitopes. Western blotting uses antibodies to identify individual proteins within a cell or tissue lysate. I can strip a single PVDF 5–6 times and still get nice signal when I re-probe.Transferring protein from gel to membrane This is by far the simplest, cheapest, and most effective stripping method I have ever used. Depending on the density of the bands, I incubate for 1–5 min. ![]() I have not had any problems with cross-reactivity in these cases.Ī3 I routinely strip PVDF membranes using 0.2 N NaOH. The only time I don't strip, but add different primary antibody, is when they are generated from different host species. Typically by this point, the membrane has about had it and I throw it away. I'll do two to four of those (stripped and re-probed), then probe for my control, which tends to give a really high signal (or some other protein with high signal). I try to strip blots of medium-low signal when you have really dense/dark bands, it's tough to fully strip all of the antibody without screwing up the membrane. The Pierce stripping buffer is nice, quick, and easy, and doesn't smell so offensive but a tris-based solution with SDS and BMe and heat works well, too (it just takes a bit longer). I regularly strip and re-probe PVDF membranes-they take the abuse better than nitrocellulose. It is also a great way to get involved with more optimization and troubleshooting (a potential headache). nonphospho-protein in different states may be difficult.Ī2 Stripping is a great way to get all you can out of a single blot. Different antigens with different sizes may be fine. It will be the same as two or multiple antibodies applied to the same blot at the same time. Re-probing without stripping can be messy. Heating with reducing agent will destroy the antibody L and H chains. Low pH dissociates the antigen-antibody complex. But I think all three methods should work the same. Is this a waste of primary in case 1, because the primary hasn't been stripped and thus is still bound? And in case 2, is this a way to simultaneously view two proteins on the same membrane? E.g., a phospho-protein and its total protein? Or does this just muddy the signals?Īlternatively, can two primary antibodies be applied to a membrane at once (in the same antibody buffer) to visualize two proteins on the same membrane?Ī1 I have not tried stripping the Western blot myself (always use new ones). ![]() Are these methods essentially equivalent in effectiveness? Also, how often can a membrane be stripped and re-probed?Īnother question: other members of the lab expose to primary, secondary, ECL, then store in water and then ![]() From the Millipore site, I've learned of three methods: their kit, low pH, and a beta-mercaptoethanol (BMe)/heat method. Q I'm new to Western blotting, and would like insight on the best method for stripping antibodies from a PVDF membrane for exposure to new antibodies. They do not represent the opinions of BioTechniques.) Mentions of specific products or manufacturers are retained from the original posts. (Posts have been lightly edited for clarity. This month, BioTechniques begins a series of excerpts, taken from some of the most recent of more than 60,000 posts. Since October 2002, the Molecular Biology Forums * have brought questioners and experts together in an online community to solve tough laboratory problems. Stripping Antibodies to Re-use Western Blot Membranes
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