Dear all,
We're doing some crystallization trials on a protein that requires 2mM
ATP in the buffer and we are having trouble measuring
reliably/reproducibly protein concentration.
This is a real problem for optimization (last screen failed because of
excessive protein concentration compared to the previous run).
What we observe:
-2mM ATP seems to prevent reliable estimation at 280nm with the nanodrop
(albeit the Akta led detector seems to do OK at 280nm) due to the major
contribution in the 200-300nm range. I guess blanking is difficult due
to the relatively large contribution of ATP vs.protein at 280
-For some reason I cannot explain, Bradford measurment (using
Pierce/Thermo dye) is also unreliable, comparable sample giving
different values. I cannot see why ATP would do tha (buffer is 20mM
Hepes, pH7.5, 150mM NaCl, 10 %Glycerol, 10% Ethylene glycol, 3mM MgCl2
and 2mM ATP).
As this is for estimation of the protein concentration while
concentrating before going into crystallization plates, the assay should
be quick (minutes) and use little amount of material (say max 5µl per
measure). An we're really interested in relative concentration (from one
experiment to the next) rather than absolute value.
I'm guessing as many of you have worked with ATPase etc, there must be a
smart way to do this.
Thanks for any input
Cedric
--
Cedric Govaerts, Ph.D.
Universite Libre de Bruxelles
Campus Plaine. Phone :+32 2 650 53 77
Building BC, Room 1C4 203
Boulevard du Triomphe, Acces 2
1050 Brussels
Belgium
