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Nuclear loading control western blot11/27/2023 Nevertheless, visualization of low abundance proteins in a cell lysate might require as much as 50 µg of total protein and at least 2 µg/mL of antibody. lysate) containing the protein of interest should be enough to be detected by a solution containing 1 µg/mL of primary antibody. In general, 1 µg of purified protein or 10 µg of a mixture of proteins (i.e. These are some useful tips to share for successful results:ġ Determine the best ratios of the target protein and primary antibodyĪlthough general guidelines for protein loading and antibody dilution are recommended by the literature and antibody manufacturers, the relative abundance of the protein of interest, as well as the titer of the antibody, used sometimes require further optimization of those parameters. 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. 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.
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