blocking for western blot
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).
pearton at u.washington.edu
"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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