Western blotting with Avidin/Biotin
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.)
Dept. of Biochemistry
Trinity College Dublin
email : crbaker at mail.tcd.ie
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