Dear Cedric, Try the BCA protein assay. We get reliable results with this, consistent with amino acid analysis. I should say that this is using the 2ml format. We do not get reliable results with the microplate method. We use 5 μl samples and our regular buffer is not too different from the one you are using.
Regards MGM Martin G Montgomery ATP Synthase Group MRC Mitochondrial Biology Unit Wellcome Trust/MRC Building Hills Road Cambridge CB2 0XY www.mrc-mbu.cam.ac.uk On 17 Jan 2014, at 14:00, Cedric Govaerts <cgova...@ulb.ac.be> 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 > > -- > 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
