Special treatment required for dried Western blots?

Hi,

So far, I always continued with incubating my Western blots in blocking
solution directly after blotting, i.e. they are still moist when I do
that.

However, if I let the blots air-dry after blotting to continue with the
treatment the next day, would I first have to specially treat the blots
in a certain way before putting them into blocking solution?

(I know that letting Western blots dry while antibody is on them makes
them unstrippable, but this is before any antibody incubation.)

Paul

I should have added that the blots are on PVDF membranes.

<[Only registered users see links. ]> wrote in message
news:1147885152.348517.148630@y43g2000cwc.googlegr oups.com...

I don't do anything special... let them dry and I just keep them wrapped to
keep them clean, then when ready just block as usual.

I store them at room temperature, and I don't know how long they'll last
like that, but certainly a week is totally fine. I think that they'll
probably last for quite sometime if you keep them dry.

Jose

[Only registered users see links. ] wrote:
Hi Paul,

I often use a quick method for PVDF westerns which uses a drying step.
After blotting, dip the membrane into methanol for a few seconds and
then allow to dry on some blotting paper either on the bench or at 37
°C. When dry you can immerse the whole membrane in your blocker PLUS
primary Ab. No additional or overnight blocking without Ab required.
The idea is that the protein on the membrane will wet first, allowing
your Ab to be soaked up by your protein of interest before there's
considerable binding of the Ab to the membrane. If you do this
procedure you will notice that the membrane is not completely wet after
the primary Ab incubation. I Incubate in blocker with primary Ab for 1
- 1.5 h, wash with TBST a few times then incubate with secondary in
blocker for at least an hour prior to colour reaction or whatever. The
whole procedure (from loading the gel to final image of blotted
membrane) takes less than a day. This is a procedure outlined in
Millipore's blotting handbook.

So, to your question about treating the membrane after drying... Just
place the membrane in blocker. The tween (or other detergent) in your
blocker will aid wetting of the protein/membrane. You may wish to do a
quick methanol wash first, however, to remove residual SDS etc. which
may allow the membrane to re-wet to quickly.

This same approach can be used to mark molecular weight bands on the
membrane without staining. Once dry, wet the membrane in about 30%
methanol. The protein will wet first, leaving opaque bands where your
abundant proteins are. Mark them with a pencil, re wet with methanol
and continue.

Many other variations on this theme available.

--
Bean

Remove "yourfinger" before replying

Thanks to everybody for the advice. I will try out the different
methods.

Paul