blocking for western blot

D. Pearton pearton at saul10.u.washington.edu
Sat Apr 22 21:40:55 EST 2000


Paul S. Brookes. (brookes at uab.edu) wrote:
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: We rountinely use 10% non-fat dry milk in TBS tween-20, for 1 hour at 37 
: degrees.  It's been our experience that blocking overnight at 4 degrees 
: requires another 1 hour at room temp' the next morning.  Our "fast" WB 
: protocol is as follows...

10%?  Wow - I get away just fine with just 2% in TBS for one hour
at room temp and get very clean backgrounds.  Then again I don't usually
use tween (it causes some of the proteins I'm interested to wash
off).  But I say give milk a try.  Any conc from 2-10% should work on
either nitrocellulose or PVDF.  The only thing I would be careful is if
you're using an anti-phospho antibody (such as the anti phospho erk, jnk,
etc) as milk contains a lot of phosphoprotein, stick to BSA for those.

: The milk we use is "Carnation" brand from Wal-Mart (no affiliation ;-p 
: ).   I think one reason this gives good results is that the time from 
: loading the gel to getting the final answer is about 8 hours, so everything 
: is still "fresh".  Even a night in the cold room probably doesn't do much 
: good for your samples.   BSA works well in our hands too (Sigma fraction V) 
: but varies between lots, so buy a big lot and stick with it.

I'm not sure that time has much effect at all.  I regularly incubate my
westerns in primary overnight at 4C and can't see much difference (signal
degradation).  I can even freeze the blot and subsequently reprobe with a
different primary (of a different species to the first) and get reasonable
detection (with no bleed-through from the fist).

Yak
--
Dave Pearton
pearton at u.washington.edu
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      "Its habit of getting up late you'll agree
         That it carries too far, when I say
       That it frequently breakfasts at five-o'clock tea,
         And dines on the following day.

The Hunting of the Snark, Lewis Carroll
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