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Sarah,

I cannot provide you with a definitive answer, but seems to me that
you could run a quick and dirty trial using hydrolyzed Fluo-3 with
slides using 1 uM Fluo-3 in buffers that range from 5.0, 6.0, 7.0,
8.0, and 9.0 (don't use amine containing buffers). Then you can
always choose some point of departure from the MP website:

http://www.probes.com/servlets/product?region=Select+Region&item=14218


I have to question your statement:

"Therefore, the fluorescence due to Calcium binding at any given
concentration will be lower at a lower pH, presumably."

When calcium is bound to EGTA and its cousins, protons are often
kicked off owing the electrostatic effects. In fact, the generation
of protons is a means used to measure the kd of many carboxylate
chelators. Thus, I would be a bit surprised if the fluorescence would
change differently when the calcium is actually bound. The kd will
change with pH, but the signal generated by bound calcium shouldn't
change much if at all.

Let us know what you find,

Mario


>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi-
>Just wondering if any of you have an idea about the pH dependence of
>fluo-3 fluorescence.  I know the Kd goes down as a result of getting
>closer to the pKa of the chelator (i.e. the on rate increases).
>Therefore, the fluorescence due to Calcium binding at any given
>concentration will be lower at a lower pH, presumably.  However, what
>I'm searching for is whether protonating one of the chelating groups
>will cause an increase in fluorescence on its own.  I'm waiting on an
>article about this from ILL, but thought I'd try you all too...
>Thanks-
>Sarah
>
>---------------------------------------------------------------
>Sarah Locknar, PhD
>Director, Neuroscience COBRE Imaging Core
>University of Vermont
>802-656-0413
>---------------------------------------------------------------


--
_________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies
FAX (510) 528-8076
1561 Posen Ave
Berkeley, CA
94706

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