![]() Increase the duration of the blocking stepīlocker was impure.PVDF requires higher concentrations of BSA than nitrocellulose PVDF requires higher concentrations of nonfat milk than nitrocellulose Nonfat dry milk, BLOTTO, blotting-grade blocker The type of membrane also affects the selection of the blocker.Ĭomparison of blocking reagents. The detection system can be optimized for minimal background with no loss of signal by testing several reagents. A variety of blocking reagents are available, including gelatin, nonfat milk, and bovine serum albumin (BSA), which are compared in the table below. Available detection methods now also include colorimetric, chemiluminescence, fluorescence, bioluminescence, chemifluorescence, and immunogold detection.įollowing transfer, unoccupied binding sites on the membranes must be blocked to prevent nonspecific binding of probes failure to completely block these sites can lead to high background. The secondary antibody can be radiolabeled, labeled with a fluorescent compound or gold particles, or conjugated to an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) for subsequent detection.įor many years, radiolabeled secondary antibodies were the method of choice for detection, but newer detection reagents have enabled less hazardous and more user-friendly methodologies, while maintaining the same degree of sensitivity. The primary antibody is specific for the protein of interest and the secondary antibody enables its detection. The steps used for immunological detection vary little and are summarized in the western blotting workflow below.Īfter the proteins have been transferred to the membrane, the membrane is blocked, incubated with a primary antibody, washed, incubated with a secondary antibody, and washed again.
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