How to Make Normal Serum Blocking Buffer

How to Make Normal Serum Blocking Buffer


Blocking is a critical step in most immunoassays, like IHC, ELISA, Westerns, etc. 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. For most assays Normal Serum Block buffers comprised of normal serum combined with a nonionic detergents is most often used.


Choosing The Host Serum Blocking Buffer 

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

*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.  

1) 1) Add appropriate amount of serum and mix with Diluent Buffer (Part # BU-117) as per Table below.

2) 2) Use immediately or store at 2-8 ºC or colder.


Serum (ml)

2 ml

5 ml

10 ml

Diluent Buffer (ml)

(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 500ml units.


About ImmunoReagents Inc.

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 immunoglobulinsprimary 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

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