western blot

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Fri Sep 23 09:10:26 EST 2005


julia_ at rocketmail.com wrote:

> Hi,
> I've being having problems with RAGE western for a while now.
> I am using the Santa Cruz antibody and it can see RAGE from the mouse
> lung and the recombinant soluable RAGE.
> I can't see any RAGE in cell culture lysates, although I can detect
> mRNA and it is been reported.
> I've been always using milk (5% in TBST) for blocking and for dilution

5% milk powder (low fat) is normaly used only for blocking, for the
incubation and washing buffers 0.1% suffices, especially in combination
with Tween. High concentrations of blocking proteins can cover the band
of interest and make it unaccessible for the antibody. On the other hand
unspecific binding of antibodies may result from leaving the carrier
protein out completely.

If milk powder does not work, try fish skin gelatin or
polyvinylpyrolidon (PVP). 

If you are using PVDF membranes (not nitrocellulose) you may also try to
dry the membrane completely and then develope the dry membrane (that is,
without the methanol step), in this case no blocking is needed because
the antibody solution can not wet the membrane, only the protein bands. 

By the way, mRNA expression does not necessarily mean protein
expression, as translation and degradation may be regulated. This is of
course the reason for assaying for both mRNA and protein.


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