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| | PP2C Activity Assay |
| Description: | HAB1 and PYL proteins were expressed and purified as described in Park et al. ((2009) Science 324(5930):1068-1071), with minor modifications. To obtain GST-HAB1, the HAB1 cDNA was cloned into pGex-2T. Expression was conducted in BL21[DE3]pLysS host cells. Transformed cells were pre-cultured overnight, transferred to LB medium and cultured at 30° C. to culture A600 of 0.5.The culture was then cooled on ice and MnCl2 added to 4 mM and IPTG added to 0.3 mM. After 16 hours incubation at 15° C., cells were harvested and recombinant proteins were purified on glutathione agarose as described in Park et al. To obtain 6×His-PYL receptor fusion proteins, expression constructs previously described by Okamoto et al. 2013, (PNAS 110(29): 12132-12137) were used. ABA receptors were expressed and purified as described above.For the values shown in Table B5, PP2C activity assays using recombinant receptors and PP2Cs were carried out as follows: purified proteins were pre-incubated in 160 μl assay buffer containing 100 mM Tris-HCl-pH7.9, 100 mM NaCl, 3 μg bovine serum albumin, 0.1% 2-mercaptoethanol, 1 mM MnCl2 with ABA or agonists (compounds of the present invention) for 30 minutes at room temperature. Reactions were started by adding 40 μL of a reaction solution containing 25 mM 4-nitrophenyl phosphate in assay buffer after which absorbance measurements were immediately collected using a 405 nm on Wallac plate reader. Reactions contained 100 nM PP2C and 200 nM PYR/PYL proteins. Some quinabactin analogs possess intrinsic fluorescence, which necessitated the use of the non-fluorescent substrate 4-nitrophenol phosphate for phosphatase assays. For inferring IC50 values, calculations from receptor/PP2C assays containing different concentrations (ranging from 1 μM to 4 nM) were. The obtained dose response data was fit to a log (inhibitor) versus response-(variable slope) model using non-linear regression to infer the IC50s, using Graph Pad Prism 6.0. |
| Affinity data for this assay | |
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