Before using antibodies to detect proteins that have been dotted or 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 or other immobilization surface 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.

Blocking Nonspecific Sites

The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal to noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding.

The proper choice of blocker for a given blot depends on the antigen itself and on the type of detection label used. For example, in applications where alkaline phosphatase conjugates are used, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase. For true optimization of the blocking step for a particular immunoassay, empirical testing is essential. Many factors, including various protein:protein interactions unique to a given set of immunoassay reagents, can influence nonspecific binding. The most important parameter when selecting a blocker is the signal:noise ratio, measured as the signal obtained with a sample containing the target analyte, as compared to that obtained with a sample without the target analyte. Using inadequate amounts of blocker will result in excessive background staining and a reduced signal:noise ratio. Using excessive concentrations of blocker may mask antibody:antigen interactions or inhibit the marker enzyme, again causing a reduction of the signal:noise ratio. When developing any new immunoassay, it is important to test several different blockers for the highest signal:noise ratio in the assay. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics.

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Overview of Western blotting

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Tech Tip #22: Determine source of nonspecific background signal in chemiluminescent Western blots.
Tech Tip #24: Optimize antigen and antibody concentrations for Western blots.
Tech Tip #32: Guide to enzyme substrates for Western blotting.
Tech Tip #67: Chemiluminescent Western blotting technical guide and protocols.

Blocking Buffer Optimization

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.

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.

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Western Blotting Handbook

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Protein Blocking Buffers
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StartingBlock Blocking Buffers
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