stripping and western blot?

D.K. dk at no.email.thankstospam.net
Sat Aug 31 16:19:19 EST 2002


"Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote:
>
>"Schwabe, TME" <tatjana.schwabe at ruhr-uni-bochum.de> wrote in message
>news:3D6F9974.9010707 at ruhr-uni-bochum.de...
>> > Seems interesting. I never heard of using low pH in Westerns, although
>it is
>> > widely used elsewhere. The only thing I wonder is how you make glycine
>> > buffer pH 1.9, when none of the conventional pH meters can accurately
>> > measure such a value? Could you post you method or the source here?
>> > Emir
>
>One can always buy ready-made glycine 1.9 or whatever from chemical
>suppliers. I use glycine 1.5-2.5 in BIAcore experiments and buy the buffers
>from BIAcore, though it is quite expensive (but !guaranteed quality! :-))
>.... Still wonder how THEY measure pH1.9. 

I might have a glaring hole in my education but this is first time in 
my life I hear about difficulties measuring pH ~ 1.5 with a properly 
calibrated pH-meter. Is there a problem? What kind? Would you 
similarly say that pH 12 is unreliable? 

With regard to the topic - yes, stripping westerns in acid with 
several thorough washes works very well (and does not strip 
bound protein as various SDS/bME protocols do). I've used
phosphate titrated to pH 1.5. As acidic proteolysis is not very 
efficient at RT, I'd expect 0.1 M HCl to do just fine too. 

DK



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