Blocking Buffers for Western Blotting

Before using antibodies to detect proteins that have been transferred to a membrane, the remaining binding surface must be blocked to prevent the nonspecific binding of the antibodies. Otherwise, the antibodies or other detection reagents will bind to any remaining sites that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking.

In practice, however, certain proteins perform better than others because they bind to the membrane more consistently or because they somehow stabilize the function of other system components. In fact, no single protein or mixture of proteins works best for all Western blot experiments, and empirical testing is necessary to obtain the best possible results for a given combination of specific antibodies, membrane type and substrate system.

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The accompanying figure illustrates the value of testing different blocking buffers as part of a Western blotting optimization experiment. In this example experiment, in which all other conditions were equal, different blocking buffers quenched or enhanced the sensitivity and specificity of the Western blot for individual proteins. In other cases, one blocking buffer or another might cause speckling or high background.

Figure 1: Importance of blocking buffer optimization. Chemiluminescent Western blot results for three proteins processed with identical conditions except for the blocking step. Each blot contains three lanes of protein corresponding to the same series of 5-fold dilutions (1:50, 1:10, 1:2). Two film exposures are shown for the fos experiment. Blocker Casein yielded the most sensitive result for Cyclin B1 protein, while SuperBlock Blocking Buffer yielded the most sensitive result for p53 and fos. In these tests involving nitrocellulose membrane, all four blockers yielded low background.