CAT assays and phosphoimagers...

batu erman erman at binah.cc.brandeis.edu
Tue Aug 1 11:49:12 EST 1995


Hi,

Recently there was a discussion about CAT elisa kits. Does anyone know if
there is a cheaper way to get these kits than to buy them from Boehringer
Mannheim? Currently they charge $275 for two plates (96X2 well) and the
reagents. This comes to about $2-3 per assay (one has to run 5 CAT protein
standards with each assay) which still is cheaper than the conventional
assays I think considering the cost of the TLC plates, the C-14
chloramphenicol, the Acetyl CoA, and other reagents, plus it saves alot of
time. But I have some worries:
1) does anyone know of any papers published with this system
2) can one get the components from other companies anti-CAT, anti-CAT-DIG,
DIG-POD, (I think I heard that a company called 5'-3' ?? also makes the
kits, does anyone have a phone number for them?)
3) also, does anyone know whether one can buy TLC plates for the
conventional assays from anybody other than Baker (through VWR) 
4) one last question, while I'm at it, does anyone know the lower detection
limit of a phosphoimager (from molecular dynamics) I recently did a serial
dilution with C-14, and quantified the spots using "grids" and here are the
numbers I got:

 2.253189e+6			1 lambda
 1.054239e+6			1/2	lambda
 1.71712e+5				1/10	lambda
 2.2535e+4					1/50	lambda
 2.7828e+4					1/100	lambda
 3.626e+4						1/500 lambda

so from this it looks like 20.000 "counts", dpm, or whatever the units for
this quantification are is the lower limit. But this is higher than some of
the spots we get when we assay reactions that give a low percent
conversion. I seem to recall molecular dynamics people saying things like
"it's linear in the full dynamic range" or something like that, and I think
we assumed that we could go as low as we wanted. Were we wrong? Has anyone
encountered this problem?

Thanks in advance for any suggestions!

Batu Erman



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