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Western blotting with Avidin/Biotin

Fergus Doherty mbxfd at unix.ccc.nottingham.ac.uk
Wed May 22 07:16:35 EST 1996

Cara Baker reported artifacual bands when probing blots with
avidin-biotinylated HRP complexes as the detection system:

> there consistently appeared two prominent bands at approx. 70-80 and 
> 120kDa molecular weight that resulted even in the absence of primary
> or secondary antibodies.


>so concluded the polypeptides
> contain a biotin moiety and hence must be mitochondrial carboxylases,
> the lower subunits of propionyl-CoA and methycrotonyl-CoA carboxylases 
> and the upper band a subunit of pyruvate carboxylase.

Could be.  I observed what I'm pretty sure are biotin containing proteins
(carboxylases) when using 125I-streptavidin, but strangely not with the ABC
system you are using.  I put this down to some kind on steric hindrance in
the ABC complex preventing binding to the biotin in the immobilised
protein.  However, you can block this binding to endogenous biotin-proteins
(if it is that).  Incubate the blot first with avidin, then with biotin. 
This forms a complex on the immobilised biotin,and biotin being monovalent
will not subsequently bind the avidin of the ABC complex.  Vector Labs have
details of this approach.

Good luck.

Fergus Doherty,
dept. Biochemistry,
University Medical School,
Queen's Medical Centre,
Nottingham NG7 2UH
Tel: (0)115 970 9366  FAX (0)115 942 2225 Internet:
Fergus.Doherty at nott.ac.uk

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