I tried to set up a malachite green assay to measure the ATPase activity of my protein and basically it seems to work but there are a few problems.
First, termination of the reaction. I could not find a detailed protocol but only what is described in a number of publications (which leaves a lot of space for interpretation of the method!!). Most descriptions said that people terminated the reaction by adding the malachite green reagent (MGR - 1:1:2:2 0.572% ammonium molybdate/6N HCl:0.232% ployvinyl alcohol:0.0812% malachite green:ddH2O), some people said they deactivated the protein with TCA. After adding the MGR I wait ~30min and then do the measurement at OD630. However, the reaction does not stop because the samples get greener and greener over time. I doubt that it's just ATP that breaks down because the "ATP only" control gives a very low OD reading and does not change over time. I also could not find anything on how people precipitate the protein before adding the MGR to stop the reaction. By adding TCA to a final conc of 10%?! And then, spinning it down and taking only the supernatent?
And second, the sensitivity of the assay. I found a whole bunch of publications were people using this assay describe the activity of their protein in the nm range (1-40ish nm). When I do my standard curve using KH2PO4, the first kind of reliable reading I get with about 100nm Pi (OD630 = ~0.04). Everything below is not significantly higher than the blank. Am I doing something wrong or is there a way to increase the sensitivity of the assay?
I also would be very thankful if someone has a protocol he/she is using in the lab and that has some details.
Thanks a lot
Edited by nimbus, 20 August 2013 - 10:15 AM.