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.

Introduction

The membrane supports, such as nitrocellulose and polyvinylidene diflurode (PVDF), used in western blotting have a high affinity for proteins. To prevent nonspecific binding of detection antibodies during the steps following transfer, unoccupied sites on the membrane surface must be blocked. Blocking buffer formulations vary widely and may contain milk, normal serum or highly purified proteins to block free membrane sites. The blocking step is imperative and improves the signal-to-noise ratio of the assay by reducing background interference. Inadequate amounts of blocker results in excessive background noise and a reduced signal-to-noise ratio; in contrast, excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the marker enzyme, which causes a reduction of the signal-to-noise ratio.

Selection of blocking buffer for western blotting applications is often system-dependent. Determining the proper blocking buffer can help to increase the system’s signal-to-noise ratio. Occasionally, when switching from one substrate to another, the blocking buffer may need to be changed in order to avoid problems with diminished signal or increased background. For example, with applications using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity. Empirically testing various blocking buffers with for use with a given system can help achieve the best possible results. No single blocking agent is ideal for every application because each antibody-antigen pair has unique characteristics. For this reason, we offer a variety of buffers to suit your western blot conditions.

Watch this video on blocking western blot membranes


Purpose and function of blocking steps

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 AP 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-to-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-to-noise ratio. Using excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the marker enzyme, again causing a reduction of the signal-to-noise ratio. When developing any new immunoassay, it is important to test several different blockers for the highest signal-to-noise ratio in the assay. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics.

Protein Detection Technical Handbook

This 84-page handbook provides a deep dive into the last step in the western blot workflow—protein detection. With a variety of detection techniques to choose from (chemiluminescence, fluorescence or chromogenic), you can select a technology to match your experimental requirements and the instruments you have available. Whether for quick visualization or precise quantitation, single-probe detection or multiplexing—Thermo Fisher Scientific offers a range of reagents and kits for western blot detection and subsequent analysis.

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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
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 Thermo Scientific SuperBlock Blocking Buffer yielded the most sensitive result for p53 and fos. Use of nitrocellulose membranes in this set of experiments resulted in low background for each blocker tested. 

Overview of available blocking buffer products

Common fixative formulations and notes on their storage and use.
Name Description †
StartingBlock Blocking Buffers Single purified protein; fast blocking; broad applicability; excellent for stripping and reprobing western blots; available in PBS and TBS with and without T20
SuperBlock Blocking Buffers Single purified glycoprotein; fast blocking; broad applicability; stabilizes plate-coated antibodies for drying; available in PBS and TBS with and without T20
Blocker BSA Blocking Buffers Purified bovine serum albumin in PBS or TBS
Blocker Casein Blocking Buffers Purified casein in PBS or TBS
Blocker BLOTTO Blocking Buffer Non-fat dry milk proteins in TBS
Pierce Clear Milk Blocking Buffer Milk proteins, clarified and stabilized in proprietary solution
Pierce Fast Blocking Buffer Proprietary proteins formulation in TBS; developed especially for rapid, 5-minute blocking of western blots as part of the Thermo Scientific Pierce Fast western blot system
SEA BLOCK Blocking Buffer Steelhead salmon serum
Protein-Free Blocking Buffers Proprietary non-protein blocking compound; available in PBS and TBS with and without T20
† PBS = phosphate-buffered saline; TBS = Tris-buffered saline; T20 = Thermo Scientific Tween 20 detergent

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Recommended reading

DenHollander, N, Defus D. (1989) Loss of Antigen from Immunoblotting membranes. J Immunol Methods 122(1):129–35.