Is immunoprecipitation (IP) required to reduce total protein load? Measure protein concentration prior to loading. Loading amounts in excess of 10 μg per lane may create conditions leading to background staining. Has the gel been overloaded with protein? Try diluting the conjugate further to reduce background. Has the antibody conjugate been titrated properly? Too much signal – signal from protein of interest obscured by high background. Has the reporter enzyme been inactivated? Or has the primary antibody been re-used, reducing concentration? We recommend preparing fresh working dilutions on day of use for both primary and secondary antibody. Has the primary antibody has been subjected to too many freeze/thaw cycles? The liberated tag may be detected as a smaller band than expected for the fusion protein.
Proteins can be subject to degradation and proteolysis during purification which may liberate an epitope tag from the protein of interest – making it impossible to detect through the epitope tag. Has the epitope tag been cleaved and lost from the protein during purification or sample preparation? If the protein of interest is very sparse, it may be necessary to enrich it in the sample using a technique such as immunoprecipitation (IP). If possible use a positive control to validate experiment. Use a Bradford assay or a spectrophotometer at 280 nm to confirm total sample protein concentration, although this does not confirm concentration of analyte. The detection limit of a Western blot is determined by a number of factors, but it is important to make sure that there is enough protein for detection in the sample prior to loading. Is there sufficient antigen present in the sample for detection? Bovine IgG may interact with the antibody due to homologous epitopes of the related species. Especially when using anti-goat or -sheep secondaries, avoid using milk or BSA in the diluent buffer. PBS/Tween 20 (0.05%) or TBS/Tween, without carrier proteins, is recommended as the secondary antibody diluent. Is the secondary antibody diluent appropriate? Increase concentration of primary and/or secondary antibody, or incubate overnight at +4oC. Is the concentration of the antibody too low? for detecting a rabbit primary use anti-rabbit secondary. Use a secondary antibody which is specific for the species the primary was raised in, i.e. If possible run a positive control for the protein of interest to confirm specificity of primary antibodyĪre the primary and secondary antibody compatible? No specific protein bands visible.ĭoes the primary antibody recognize the protein of interest?Ĭheck with the manufacturer of the primary antibody that it has been validated for detection of protein or epitope tag of interest by Western blot.
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