Posted by
Jurkevic, Aleksandr on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Asante-Red-Calcium-tp6746843p6755599.html
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Thomas,
AM conjugate of ACaR has QY 0.03-0.04 and molar extinction coef. 23,000. It may explain to some extend low brightness of this indicator. For comparison, fluo-4 has QY 0.14 and molar extinction coef. of 77,000. Unfortunately, fluor-4 is non-ratiometric...
We were interested in a dextran conjugate of ACaR, but the company told us that it will be available only next year.
Aleksandr
Aleksandr Jurkevic, PhD
Associate Director
Molecular Cytology Core
University of Missouri-Columbia
120 Life Sciences Center
1201 E. Rollins St.
Columbia, MO 65211-7310
Phone: 573-882-4895
Fax: 573-884-9676
website
http://www.biotech.missouri.edu/mcc/-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Thomas Volksdorf
Sent: Friday, September 02, 2011 2:10 AM
To:
[hidden email]
Subject: Re: Asante Red Calcium
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Hello Jason!
Like any other AM-dye (at least as far as I know...), the AM-ester of the
ACaR is supposed to be hydrolised automatically by intracellular esterases,
so after you have incubated the cells with the dye you let them stay for
another 45-60 minutes and then the dye should be ready. So it says in the
datasheet, and the few experiments that have been done by other researchers
followed this procedure.
I also followed another procedure published by invitrogen to test the dye
for responsiveness - basically you hydrolise the ester in KOH. After that I
was able to get a signal from my solution, and the signal @ 650 nm increased
when adding calcium, so the dye itself works.
So at least theoretically the AM-ester shouldn't be a problem, but thank you
very much for trying to figure out why my signals aren't as good as they are
supposed to be :)