Try preparing your Bradford standard curve with your protein buffer (including the ATP). This is how I reliably get around detergent and other compounds that interfere.
Hope this helps, Kelly ******************************************************* Kelly Daughtry, Ph.D. IRTA Fellow Mechanisms of Mutation Group Molecular Genetics Laboratory, MD E-301 National Institute of Environmental Health Sciences 111 TW Alexander Drive Research Triangle Park, NC 27709 Tel. (919) 541-3452 ******************************************************* On Fri, Jan 17, 2014 at 9:00 AM, 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 5痞 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 >
