reprobe western blots

Tom Thatcher ttha at uhura.cc.rochester.edu
Thu Jan 5 21:55:07 EST 1995


In <anon-0501951543180001 at hammer.biochem.uci.edu> anon writes:

>In article <3eg2t9$8hl at neuro.usc.edu>, william at neuro.usc.edu (Fiberman) wrote:

>> Hello, Netters,
>> 
>> Can a western blot filter be "reprobed" like a Southern blot filter?  If
>> so, what is the best way to strip the original antibody?  I am using a
>> secondary antibody conjugated to alkaline phosphatase, and visualizing
>> with addition of color substrates NBT and BCIP.  I would appreciate your
>> response.
>> 
>> -fm

>   I think this method causes a precipitate to form, and you can't strip
>and reprobe when using this method.
> 

You could reprobe with a second antibody (to a different protein) without
stripping as long as you used primary and secondary antibodies with
different specificities

eg.  Ab#1 from rabbit<<goat-anti-rabbit-alk phos
     Ab#2 from mouse<<goat-anti-mouse-alk phos

You need before and after pictures to know which bands are new.  Or, use
a second step that causes a different color reaction.

You can strip the Abs with the same stuff you use to strip affinity columns
(low pH glycine, etc.) but you won't get rid of the first color bands
themselves.

-- 
Tom Thatcher                          | You can give a PC to a Homo habilis,
University of Rochester Cancer Center | and he'll use it, but he'll use it
ttha at uhura.cc.rochester.edu           | to crack nuts.



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