Avidin/biotin blocking

From:Gayle Callis

We bite the bullet and routinely block for endogenous biotin in ALL tissues
when we stain with Avidin or Strepavidin methods. It just easier to apply,
and we don't find it that expensive. 

Vector just released a new Strepavidin/Biotin block (for people who use
Strepavidin, and there are some considerations about this.  See following
comments from Vector Tech Services 
Streptavidin does have some subtle binding differences
than avidin. Streptavidin has been shown to bind to fibronectin receptors
that are expressed on cell types. These integrins (extracellular matrix
receptors) mediate cell adhesion, migration etc and streptavidin binds to
these too. So the bottomline this that streptavidin based detection systems
may give background/non-specific staining due to this inetgrin binding that
is not blocked by a standard avidin/biotin blocking step. Hence the idea
behind the streptavidin/biotin kit. See the following reference:

Alon, R. et al (1991) Cell-adhesive properties of streptavidin are mediated
by the exposure of an RGD-like RYD site. Eur. J. Cell Biol. 58:271-279.

The nomenclature in this article is dated. The GpIIbIIIa is now known as the
alpha 5 beta 1 dimer that specifically binds fibronectin via the RGD amino
acid sequence. There are antibodies available against the alpha 5 subunit
itself. Migratory cells, such as embryonic cells, some tumor cells etc would
be high expressors of this integrin. Obviously you could either block with
the streptavidin kit or use an avidin based detection system.


If it is an expense consideration, you can make up your own avidin/biotin
blocks (I have recipe)  - and even add these to your normal serum block
(NSB) instructions are in Vector kit on how to do this.  We routinely do
NSB/avidin, rinse, then NSB/biotin, rather than add the biotin block to
primary antibody.  

Jules Elias once commented to me privately - if I can live with level of
endog biotin seen without doing a block and this amount of background
doesn't mess up seeing what I'm are detecting with my antibody, then I
shouldn't bother with endog biotin blocking. So it became our choice, we
decided to block on all tissues.  

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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