stripping and western blot?

Emir Khatipov khatipovNO at NOuchicago.edu
Tue Sep 3 11:54:24 EST 2002


"D.K." <dk at no.email.thankstospam.net> wrote in message
news:akufm4$fui$1 at news.doit.wisc.edu...
> ffrank at rz.uni-potsdam.de (Frank =?iso-8859-1?q?F=FCrst?=) wrote:
> >dk at no.email.thankstospam.net (D.K.) schrieb:
> >
> >> 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?
> >
> >At high pH you get an error because the electrode will respond to Na+ or
> >Li+ (it's called "Alkali-Fehler" in german, "alkali error" or something
> >like that). But I also have never heard of an error at low pH.
>
> Yes, this effect is easy to understand. Although I doubt it is significant
> at pH 12 - IIRC, most electrodes' specs claim linear response to 12.
>
> DK

Don't get so emotional, Dima! I was just trying to get the point about the
accuracy knowing how most people in molbio labs would calibrate pH meters -
by one buffer (pH 7) !. I also noticed that most labs buy less expensive pH
meters that cannot be easily calibrated over the whole pH range. For
example, if you calibrate the instrument with buffers pH 7 and pH 4 and then
check the reading of the buffer pH 10, you would not get the correct reading
of 10. The opposite is also true: calibrating with pH 7 and pH 10 does not
allow correct reading at pH 4. Calibration with 3 buffers does not work at
all for the same reason. You should try that, and if it works in your hands,
I would be happy if you help me to close the gaping hole in my education
:-). Most pH meters have (claimed) response range of pH 1-12. Thus, given
that pH 1.9 is close to the extreme and the above considerations of
unreliable calibration, I don't believe people get paranoid enough making
sure that the pH they get is in fact 1.9. And they don't, luckily it is not
in fact SO critical in many cases, including Western. As a result, pH 1.9 is
pH 1.9 at a certain pH-meter in a certain lab. And Tatjana admitted that.
However, in e.g. BIACore pH is very important: a difference in 1/10 of pH
may disrupt some binding surfaces and result in baseline drifts, etc. In
addition, you might have a good pH meter, but not so good electrode that
would not have such a wide linear response range...

I personally don't think this thread should steer away from the initial
topic though. I was personally happy to learn that stripping can be done
with acids, and wonder why, if it works so good, few labs know about this
method and instead prefer stinking up everything with bME.

Cheers,
Emir





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