The solution might be as simple as measuring absorbance at 290 nm. We routinely measure concentrations in the presence of 100 uM ATP ( usually without concentrating though). That said 2 mM ATP is a lot more than we usually deal with and may interfere even at 290 nm. The interference from ATP drops off quickly above 280 nm, so you may have better results at slightly higher wavelengths.
One can do a better estimate if needed, but the extinction coefficient at 290 nm should be near 0.5 - 0.65 times the extinction coefficient at 280 nm. The exact numbers will depend on how many tryptophans vs tyrosines your protein contains, and could be sorted out at lower concentration. This should allow rapid detection using a nanodrop instrument. Shae Padrick On Jan 17, 2014, at 8:10 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 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 ________________________________ UT Southwestern Medical Center The future of medicine, today.
