Western blot

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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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. 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.

Importance of 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.

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.

Blocking buffer selection table

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Cat #Blocking BufferELISAWestern BlotDot BlotImmunohisto-chemistryDNA/RNA Hybridization
37538StartingBlock (PBS) Blocking Buffer

37542StartingBlock (TBS) Blocking Buffer

37539StartingBlock T20 (PBS) Blocking Buffer

37543StartingBlock T20 (TBS) Blocking Buffer

37515SuperBlock Blocking Buffer (PBS)

37535SuperBlock Blocking Buffer (TBS)

37517SuperBlock Blocking Buffer – Blotting in PBS 

37537SuperBlock Blocking Buffer – Blotting in TBS 

37516SuperBlock T20 PBS Blocking Buffer

37536SuperBlock T20 TBS Blocking Buffer

37527SEA BLOCK Blocking Buffer

37520Blocker BSA (TBS)

37525Blocker BSA (PBS)

37532Blocker Caesin (TBS)

37528Blocker Caesin (PBS)

37530Blocker BLOTTO (TBS)

37570Protein-Free (TBS) Blocking Buffer

37571Protein-Free T20 (TBS) Blocking Buffer

37572Protein-Free (PBS) Blocking Buffer

37573Protein-Free T20 (PBS) Blocking Buffer

37576Pierce Fast Blocking Buffer 

37587Pierce Clear Milk Blocking Buffer 

T2015I-Block Protein-Based Blocking Reagent

00-0105Membrane Blocking Solution