double immunoblotting

Esk sophiemalpass at
Sat Jan 11 13:36:05 EST 2003

On Fri, 10 Jan 2003 21:11:05 +0000, Jason Wong wrote
(in message <a07dedf1.0301101311.6761385e at>):

> I'd like to try my hand at simultaneous primary antibody incubations
> in my Western immunoblots.  My bands are separated sufficiently in a
> 12% gel, and stripping doesn't seem to work.  So I figured I'd just
> stain for both the protein of interest (SNAP-25) and a housekeeping
> protein (B-actin) at the same time.  I have heard from others on this
> group that many people do this, but does anyone have a good paper they
> can use to site this method?  I've tried to find one from Pubmed and
> Ovid but I'm coming up a bit blank.

The first obvious thing to do would be to run 2 lanes of your protein side by 
side. cut the membrane up, label each separately. they can then be lined up 
side by side later! as long as you have enough protien. Careful use of cut 
corners of the membrane to ensure you get them all lined up correctly is 
needed, and i rather like coloured markers such as bio-rad kaliedascope, as 
you can see them on the membrane really nicely.

If you do want to incube both together, just make sure you buy b-actin from 
the same animal as your primary, and ensure you've got both working nicely on 
their own, in case anyone quizzes you on specificity.

bear in mind if you're using ECL etc detection, you'll also have a challenge 
getting the bands for the two antibodies to come out at the same intensity 
with the same secondary ab  concentration.


Sorry if this get s posted multiple times . newbie.

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