Membrane stripping after HRP-conjugated streptavidin incubation

Emir Khatipov khatipovNO at NOuchicago.edu
Thu Feb 7 12:35:09 EST 2002


Streptavidin-biotin interaction is very strong and AFAIC cannot be disrupted
completely, even under harsh conditions, like high or (particularly) low pH.
You may still be able to "bleed" some of the bound SA-HRP by soaking the
membrane in a low pH buffer (pH 1 - pH 2). However, there is no guarantee
that your antigen will sustain that punishment. Thus, I would try short-term
washings (1-5 min) first. Another problem is how you will estimate the
amount of biotin being freed, which is probably by HABA assay or by
reprobing with a different SA-conjugate (alkaline phosphatase?, fluorescent
probe?).
It is also known that avidin-biotin interaction is weaker than that of
SA-biotin. Thus, I would suggest trying avidin-HRP (check the catalogs; I
guess Sigma has one).
Also check Meth Enzymol v.184 (1990) that has reviews on SA-avidin
applications.
I did not try the above myself. It is just an "educated" suggestion :-).
Emir


"Patrice DK" <pb at nordicbioscience.com> wrote in message
news:3c62b151$0$10684$4d4eb98e at news.dk.uu.net...
> Hi all,
>
> I wonder if there is a way to strip a nitrocellulose membrane after it was
> incubated with HRP-conjugated streptavidin for detection of biotinylated
> proteins. I would like to re-incubate my membrane with a fresh
> HRP-streptavidin since I strongly suspect the previous one to be out. As
> streptavidin-biotin interaction is very strong, Í think a re-incubation of
> the membrane with the new one won't help so much since all biotin sites
are
> probably already saturated.
> Does someone have an idea about that or has tried to get rid of
> HRP-streptavidin,
> Many thanks for your help,
>
> Patrice
>
>





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