Cedric,
I suspect you are correct in that the 2 mM ATP, which will have
significant absorption at 280 nm, is contributing a high background
absorbance. The Bradford method is notoriously idiosyncratic, as it
depends on hydrophobic content of the protein, and is affected by
substances that change the gross hydrophobicity of solution. The
glycerol and/or EG may be a factor here. Glycerol is going to interefere
with the Lowry-type assays as well.
If your protein is a metalloprotein with a stoichiometric amount of
metal ion, e.g. a zinc-metalloenzyme, then by far the best way to go is
to do ICP-OES or TXRF for the metal ion.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 1/17/2014 9:00 AM, Cedric Govaerts wrote:
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
