Serum Blocking Buffers: ELISA’s/Westerns
Serum Blocking Buffers are used frequently in immunoassays and are a critical step. Blocking “fills-in” the unoccupied spaces of the solid phase that are not occupied by immobilized proteins. Blocking prevents non-specific binding of the antibodies. Without blocking agents, the antibodies could bind without specificity and lead to false signaling and/or background issues. While there is no single “best” blocking agent, empirical testing is needed to validate which methods works best in each assay. Blocking shoud be done for the following assays: ELISA, Western Blotting, Immunohistochemistry, etc.
*IR Tip- In general, a mixture of proteins combined with nonionic detergents is most often used.
Choosing the Correct Blocking Serum
When choosing which normal serum to use, it is recommended to use serum that is derived from the same host species as the secondary antibody. For example:
Secondary Antibody = Donkey anti-Rabbit IgG-HRP
Blocking Serum = Donkey Serum
*IR TIP-CAUTION: Do not use serum from the same species as the Primary Antibody, as this will compete for sites with the secondary antibody and reduce the signal.
How to Prepare 5% (v/v) Serum Blocking Buffer
Many immunoassays use a 5% (v/v) Serum Blocking Solution in Phosphate Buffered Saline (PBS) with Tween® 20 detergent.
- Add serum and mix with Diluent Buffer (Part # BU-117) as per table below.
- Use immediately or store at 2-8 ºC or colder.
|Serum||2 ml||5 ml||10 ml|
|Diluent Buffer (Part # BU-117)||38 ml||95 ml||190 ml|
|Total Volume||40 ml||100 ml||200 ml|
*ImmunoReagents supplies serum in 2, 5, and 10 ml units. Serum Diluent Buffer is supplied in 500 ml units.
ImmunoReagents offers a variety of serum for your immunoassay. Our most popular are:
Normal Donkey Serum *IR-Tip Donkey Serum is a good universal blocking agent. Not to be used if Primary antibody is from Donkey host.
ImmunoReagents, Inc. is a leading global manufacturer of quality antibodies and reagents used in pharmaceutical research, life science research and in vitro diagnostics. Product offerings include a wide range of immunochemistry reagents such as purified immunoglobulins, primary antibodies, and secondary antibodies covering a broad spectrum of immunoglobulins from various species. ImmunoReagents also provides custom manufacturing to meet specific customer requirements while adhering to cGMP guidelines and ISO quality systems requirements. The company is located in Raleigh, North Carolina