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Blocking biotin labeled blots with BSA?

Blocking biotin labeled blots with BSA? - Protocols and Methods Forum

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Old 01-10-2007, 11:21 PM
WS
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Default Blocking biotin labeled blots with BSA?



Dear experts,

I hope I do not have a problem:

Intending to detect biotin derivatized proteins on PVDF membranes, I
have electroblotted my sds gel on the membrane and incubate it with the
biotin reagent o/n now. The next step would be blocking the membrane as
i usually do for western blotting with a buffer containg BSA.

I suddenly started worrying if BSA (0.5%, together with 0.02% TX100 in
buffer) is really suitable for this as I am afraid (fears are supported
by my beloved wife who is an expert, too (fortunately, of course ;-)
that I might introduce lotsa biotin and get strong background. My
worries are enlarged by a reference where the authors used "low biotin
containing BSA" from Aldrich, which I however cannot find in the
sial.com online catalogue. The original protocol I am cooking refers to
a kit with an undisclosed blocking agent.

As I do not want to waste my samples, I am asking you for urgent help.
My BSA is ordinary fraction V. Do I need to worry? What could I use
instead? On the shelf-stuff is preferred (ordinary gelatin is
available). Is there a way to kill biotin present in BSA by chemistry -
oxidation of the sulfur with H2O2 maybe?

Next step will be incubation with streptavidin-peroxidase / ECL. quite
sensitive, hopefully / unfortunately.

Appreciating your input!

Wolfgang

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