Nitrocellulose membrane is more fragile but less expensive than PVDF, and does not require pre-wetting with methanol or cause as much non-specific binding. Though more expensive, the PVDF membrane is stronger and has a high absorption capacity with proteins, which makes it suitable for CBB staining, detection with certain substrates for alkaline phosphatase, and re-probing. Nitrocellulose and PVDF membranes are commonly used. Comparison between blotting apparatuses Membrane A semi-dry or a tank system can be used for transfer. Proteins separated by SDS-PAGE are “transferred” from the polyacrylamide gel to a membrane, using a specialized apparatus (blotting apparatus). The principle and method of polyacrylamide gel electrophoresis (SDS-PAGE).The following sections describe the procedure starting from electrotransfer of proteins to the membrane.įollow the links below for the methods used to generate antibodies, and the principle and method of SDS-PAGE. The bound enzyme activity is used to detect the target protein and visualized by a chemiluminescent or chromogenic method. The membrane with transferred proteins is then probed with a primary antibody (an antibody specific for the target protein), washed, and reacted with a secondary antibody labeled with an enzyme, such as horseradish peroxidase (HRP). In Western blotting (WB), target proteins are transferred to a hydrophobic membrane after SDS-PAGE and detected using specific antibodies.Īfter SDS-PAGE, a membrane is placed on the gel, to which the separated proteins in the gel are electrophoretically transferred. FAQs – In Vivo Isotype Control AntibodiesĪdvanced MHC Tetramers and Antibodies Searchįind products for Western blot The principle.Protein-Protein Interaction Detection Technology.FGF-Max (Recombinant human FGF-1/FGF-2 chimera).Afamin/Wnt3a CM and recombinant protein Show submenu.Non-Classical MHC Tetramers Show submenu.MHC Class I & Class II Tetramers Show submenu.
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