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 Buffer ELISA Western Blot Dot Blot Immunohisto-chemistry DNA/RNA Hybridization
37538 StartingBlock (PBS) Blocking Buffer

37542 StartingBlock (TBS) Blocking Buffer

37539 StartingBlock T20 (PBS) Blocking Buffer

37543 StartingBlock T20 (TBS) Blocking Buffer

37515 SuperBlock Blocking Buffer (PBS)

37535 SuperBlock Blocking Buffer (TBS)

37517 SuperBlock Blocking Buffer – Blotting in PBS  

37537 SuperBlock Blocking Buffer – Blotting in TBS  

37516 SuperBlock T20 PBS Blocking Buffer

37536 SuperBlock T20 TBS Blocking Buffer

37527 SEA BLOCK Blocking Buffer

37520 Blocker BSA (TBS)

37525 Blocker BSA (PBS)

37532 Blocker Caesin (TBS)

37528 Blocker Caesin (PBS)

37530 Blocker BLOTTO (TBS)

37570 Protein-Free (TBS) Blocking Buffer

37571 Protein-Free T20 (TBS) Blocking Buffer

37572 Protein-Free (PBS) Blocking Buffer

37573 Protein-Free T20 (PBS) Blocking Buffer

37576 Pierce Fast Blocking Buffer  

37587 Pierce Clear Milk Blocking Buffer  

T2015 I-Block Protein-Based Blocking Reagent

00-0105 Membrane Blocking Solution