Tips for Step 8: Block Non-specific Binding Sites

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Overview

To prevent non-specific antibody binding to tissue surface structures such as Fcγ receptors, blocking is traditionally performed prior to incubation with the primary antibody (Kim et al. 2016).

Although the impact and merit of this blocking step has recently been questioned by Buchwalow et al. (2011), we recommend including a blocking step in your experimental design.

Blocking advice:

• Block with serum from the same species as the tissue; the immunoglobulins present in the serum will bind to the receptors present on the tissue


• Alternatively, use normal serum from the same species as the one in which the secondary antibody was generated


• Block with 10-20% normal serum


• Never block with normal serum from the same species that the primary antibody was generated in. This could lead to blocking of reactive sites or higher background


• If serum is unavailable, use bovine serum albumin, non-fat milk or gelatin


• Do not block with milk when using biotin-avidin detection systems. Milk contains biotin and therefore will result in staining artifacts/high background (Kim et al. 2016)