Why 3H-GTP in GTP-binding assay?

Bin Lin binlin at umich.edu
Sat Jun 28 11:53:29 EST 1997


Hello all:

I am a graduate student in the Dept. of Biology at University of
Michigan (Ann Arbor) studying a GTP-binding protein in Caulobacter
cresentus. Now I am thinking to measure the GTP binding as well as GDP
dissociation constant of that protein.

After reading many papers about in vitro GTP binding assay, I find that
all most all the experiments were done using 3H-GTP(GDP), instead of
other radioactive isotopes like 32p-GTP, 35S-GTP etc., to calculate the
ligand binding constant. There must be some  crucial reasons behind it.
Might it be for the sake of half-life (then why not 14C-GTP?), LS
counting accuracy (but you have to convert cpm to dpm because of the
scintillation
quench of low energy particles.), data repetition, or even the purity of
the commerical radioactive nucleotide ...? 

Some people say it's safer to use 3H than to use other isotope. But in
my point of view, mistakes in dealing with 3H might cause more trouble:
it's hard to be detected! So, personally I prefer using 32p or 35S if
they
don't affect the data accuracy. 

It would very appreciated if any of your experienced people could give
me some comments about that!

Thanks in advance!

Bin Lin
binlin at umich.edu



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