| Assay Method Information | |
| | Malachite Green ATPase Assay |
| Description: | (1) Greiner 384-well (Greiner 781101) or Costar 384-well flat-bottomed polystyrene multiwell plates (VWR). (2) Assay buffer of (a) 100 mM Tris-HCl, pH 7.4, (b) 150 mM KCl, (c) 6 mM MgCl2. Stored at room temperature. (3) 0.0812% (w/v) malachite green (M 9636, Sigma Aldrich Ltd., Poole, UK). Stored at room temperature. (4) 2.32% (w/v) polyvinyl alcohol USP (P 1097, Sigma Aldrich Ltd, Poole, UK) in boiling water (see Comment 1), allowed to cool, and stored at room temperature. (5) 5.72% (w/v) ammonium molybdate in 6 M hydrochloric acid. Stored at room temperature. (6) 34% (w/v) sodium citrate. Stored at room temperature. (7) 100 mM ATP, disodium salt, special quality (47699, Sigma Aldrich). Stored at −20° C. (8) E. coli expressed yeast HSP90 protein, purified >95% (see, e.g., Panaretou et al., 1998) and stored in 50 uL aliquots at −80° C. Method 1. Dilute test compounds to 500 μM in AR water (DMSO concentration will be 2.5%). Transfer 2.5 μl of these compounds directly from the daughter plate to the assay plate, giving a final assay concentration of 100 μM. To obtain 12 point IC50 values, perform serial dilutions 1:2 to produce a range of assay concentrations from 100 μM to 97.6 nM (2.5% DMSO), and transfer 2.5 μl of each concentration into the assay plate. Column 1 in the assay plate contains no compound, as a negative control. An additional row with no compound is also used as a background. 2. Prepare ATP by diluting 100 mM stock to 925 μM with assay buffer, and aliquot 5 μl of diluted ATP to each well including controls (final assay concentration 370 μM). 3. Add 5 μl of buffer to background row. 4. Dilute enzyme preparation to 1.05 μM with assay buffer, and aliquot 5 μl into each compound well and to the negative control column. 5. Collect the reagents to the bottom of the well, cover plate with plate seal and incubate overnight at 37 deg C. 6. First thing in the morning prepare the Malachite Green Reagent. Add 2 parts of Malachite Green Solution, 1 part of Polyvinyl Alcohol Solution, 1 part of Ammonium Molybdate Solution, and 2 parts of AR water. 7. Invert to mix, and leave for approximately 1 hour until the colour turns from brown to golden yellow. 8. Add 40 μl of Malachite Green Reagent to each well, allow 5 mins for colour to develop. 9. Add 5 μl of Sodium Citrate Reagent to each well (see comment 2) 10. Re-cover with plate seal and shake on plate shaker for at least 15 mins. 11. Measure Absorbance at 620 nM using a suitable plate reader (e.g. Victor, Perkin Elmer Life Sciences, Milton Keynes, UK). Under these conditions, the control absorbance is 0.9 to 1.4, and the background is 0.2-0.35 giving a signal to noise ratio of 12. The Z′ factor calculated from data obtained using these conditions is between 0.6 and 0.9. Comments |
| Affinity data for this assay | |
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