If not already included, add up to 0.01–0.Consider blocking overnight at 4✬ or at least 1 hour at room temp (increase length of incubations if necessary).Increase the concentration of blocking reagent (e.g., BSA, nonfat dry milk, etc.) from 5% to 7% (w/v).Insufficient blocking of nonspecific sites Increase the Tween-20 concentration to 0.01–0.5% (v/v).Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding.Include progressively stronger detergents in the washes for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween-20.NOTE: Depending on the secondary antibody that is used, 55 kDa and 27 kDa heavy and light IgG chains, respectively, of the primary antibody may be detected. Continue with electrophoresis and immunoblotting as described under Western blotting procedure in protocol 1. Try PBS-T instead of TBS-T (do not do this if using phosphospecific antibodies) Load up to 510 l of sample per 1.0 mm well width for gels of 0.75 mm thickness.Increase the salt concentration of your TBS-T.Increase the ionic strength of the incubation buffers.Nonspecific interactions occurring due to ionic associations for example, avidin, a glycosylated protein, may bind to more acidic proteins on blots Blot native proteins as a comparison, e.g., by blue native PAGE.Compare the binding of other monoclonal or polyclonal antibodies.Monoclonal antibodies reacted nonspecifically with SDS-denatured proteins Use purified IgG primary antibody fractions and affinity-purified blotting-grade cross-adsorbed secondary antibody.Primary or secondary antibody contaminated with nonspecific IgG or with IgG cross-reactive among species Check research literature for existence of isoforms or variants.Protein exists in several different isoforms Repeat immunodetection with secondary antibody alone to check for nonspecific binding.Use an affinity-purified secondary antibody.Decrease or optimize the concentration of the secondary antibody, e.g., using a checkerboard screening protocol.Secondary antibody concentration too high, leading to nonspecific binding Check antibody specificity with a blocking peptide (pre-incubate the antibody with an excess of the same sequence used to generate the antibody see Demonstrating Antibody Specificity).Bio-Rad now offers PrecisionAb™ Validated Western Blotting Antibodies for superior performance in western blot detection Optimize primary antibody concentration.Use an affinity-purified primary antibody.Primary antibody concentration too high or cross-reactivity with similar epitopes on other proteins Multiple nonspecific bands on the blot may be due to antibodies of poor quality or at too high a concentration, insufficient blocking, or nonspecific binding due to the presence of SDS.
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