Western blotting with Avidin/Biotin

crbaker crbaker at mail.tcd.ie
Wed May 22 05:08:37 EST 1996


During the course of trying to characterise anti-peptide antisera 
raised in the lab we have come up with a rather irritating artifact.

To maximise the sensitivity of our westerns we started to use the 
Vectastain ABC kit whereby the primary antisera is recognised by a 
biotinylated secondary immunoglobulin and the blot incubated with
a mixture of Avidin DH and biotinylated horse radish peroxidase. 
Following washing, blots are developed using the ECL system. 
However when probing human platelet and rat synaptosomal membranes 
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. We have supposed these species must be some
sort of avidin binding polypeptides. As lectins have the ability to 
recognise the terminal mannose side chains in avidin we tried to block
with alpha-methyl-D-mannoside but to no avail 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.
An extensive high salt washing of the membranes did not deplete the 
membranes of the bands.
After much tinkering, we have now changed to HRP-labelled secondary
antibody and wanted to know if anyone else has had similar experiences
or suggestions as to the cause.
(No affiliations etc.)

Cara Baker
Dept. of Biochemistry
Trinity College Dublin
email : crbaker at mail.tcd.ie 



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