However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. In general, the smaller the protein the faster it migrates through the gel. Western blotting is a technique that separates proteins based on size. Why is the actual western blot band size different from the predicted? The optimal antibody concentration must be determined empirically. Typical antibody concentration range = 0.2 - 5.0 ug/mL, or begin with a 1:1000 dilution of antibody reconstituted at 1 mg/mL. ~25 ug/lane = Amount Cell/Tissue Lysate Protein Used Per Lane Efficient protein of interest transfer is dependent on 1) protein size, 2) transfer power settings, and 3) transfer time.More/longer washing steps can help reduce both specific and nonspecific antibody staining.Always run a positive control to ensure antibody activity (purified protein, transfected lysate, etc.).Increasing/decreasing enzyme substrate incubation time can help amplify/reduce signal, respectively.Increasing/decreasing primary antibody concentration can help amplify/reduce signal, respectively.Western Blotting / Immunoblotting (WB / IB) General Tips to consider when performing a western blot: IP Tips & Tricks | Immunoprecipitation (IP) Protocol.ELISA Tips & Tricks| Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol.ICC Tips & Tricks | Immunocytochemistry (ICC) Protocol.IHC Tips & Tricks | Immunohistochemistry (IHC) Protocol.
WB/IB Tips & Tricks | Western Blotting/Immunoblotting (WB/IB) Protocol.High Throughput Antibody Characterization Services.Loading Control Antibodies for Western Blot.Enhanced Validation Polyclonal Antibodies.
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