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In fact if you neutralize any acidic buffer such as Pi or MOPS
with NH4OH instead of NaOH or KOH, then equilibrate with reservoir
containing the same buffer at a different pH, the pH will tend
toward that of the reservoir. Ammonia diffuses as the gas NH3,
converting NH4MOPS to HMOPS, titrating the buffer to greater HMOPS/MOPS-
ratio. Vapor diffusion continues until activity of NH3 in the
drop is the same as the reservoir which will be achieved when
the pH is the same as the reservoir if all else is the same,
because the same pH ensures the ratio of NH4MOPS
to total MOPS and the ratio of NH3 to NH4 is the same.
However if you seal your wells with a tape which is significantly
permeable to NH3 gas, the whole system will drift slowly acid
and you will need to harvest and freeze the crystal before
that goes too far.
Better to adjust the buffer with KOH, take out some for
the drops to which add a little NH4OH to make it slightly more
basic, and keep the protein from precipitating when mixed.
Then gradually remove the NH3 by vapor diffusion to the
reservoir or through the tape, and the pH will drop slowly
to that of the reservoir and stay there.
Now if you want to go the other way you might use CO2
instead of NH3 as the volatile constituent, which brings
us back to an old thread about perrier water or even
soda pop as precipitant. Heck, if you just want to lower
the pH gradually while retaining osmotic balance, replace
your reservoirs with 0.5 ml coca-cola and leave it sealed
overnight.
Ed
[EMAIL PROTECTED] wrote:
I think a low-pH well buffer with volatile components (say (NH4)acetate
) would do the job, although I don't know about speed. Maybe you could
measure it with some old-fashioned pH sensitive dye in a trial drop?
Phoebe
At 01:08 PM 12/4/2006, Andrew Wong wrote:
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Hi
Just a quick question.. for doign hanging drop, is there anyway that we
can decrease the pH of the drops on a (somewhat) controlled way, say over
a period of couple of weeks? Thanks
Andrew
---------------------------------------------------------------------------------------------------------------------------
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/index.html
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
