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| | ATP-Biotin Probe Labeling and Inhibitor Competition |
| Description: | The ATP-biotin assay was performed in buffer containing 25 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, and 2% DMSO. Purified hKSR2-rMEK1 was assayed at 0.5 μM. In particular, hKSR2-rMEK1 was pre-incubated with 20 μM of the indicated compounds for 15 minutes. Then, ATP-biotin (Pierce Cat. #88310) was added to a final concentration of 2 μM. Reactions were incubated at room temperature for 5 minutes before being stopped by the addition of 6×SDS loading dye. Samples were then electrophoresed on a 4-15% Tris-HCl SDS gradient gel, transferred to nitrocellulose membranes and blotted with Strepdavidin-HRP. Enhanced chemiluminescence signals corresponding to labeling on KSR2 and MEK1 were visualized and quantified using the Biodoc system (Biorad). Relative signals of Streptavidin HRP on bands corresponding to KSR2 and MEK1 in the presence of compounds relative to DMSO controls were used to determine Percent Inhibition of ATP Probe Labeling . Control experiments using ATP as a competitor were used to determine that the ATP-biotin probe specifically labels the active-site of KSR2 and MEK1. Representative results of the ATP-biotin assay are shown in FIGS. 1-2.ATPbiotin transfers a desthio-biotin group via a reactive acyl-phosphate linkage onto active-site lysines when bound to either KSR2 or MEK1 (Patricelli et al, Biochemistry, 2007, 46:350-358). It was confirmed that this label leads to a covalent attachment of desthiobiotin on KSR through detection with Streptavidin-HRP and intact mass spectrometry, the latter of which confirmed the addition of a two or one desthiobiotin groups (equivalent to a mass increase of 196.1 Da per desthiobiotin group) onto both KSR2 and MEK1, respectively, under non-saturating conditions. Without being bound by theory, competition experiments with free ATP suggested that ATPbiotin labeling occurred within the active sites of both KSR2 and MEK1, as shown in FIG. 3, and free ATP IC50 values of 86 μM and 133 μM, respectively, were measured as shown in FIG. 4. Results of the ATP based competition experiments support the utility of this assay for identifying direct binders of KSR2, MEK1, or both kinases within purified complexes.For the ATPbiotin labeling competition screen, samples were applied to a 4-20% Tris-HCl gel, separated, and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin diluted in TBS-T for 30 minutes and subsequently probed with the Pierce High Sensitivity Streptavidin-HRP. After several washes, the membranes were visualized using enhanced chemiluminescence on a Biodoc (Biorad). IC50 values were determined under similar assay conditions with slight modifications: 0.1 μM KSR2:MEK1 was pre-incubated with a dose-range of compounds (27 nM to 20 μM in three-fold dilutions prior to the addition of ATPbiotin. Assays were quenched and analysed similarly to as described above. Sigmoidal dose response curves were used to derive IC50 values in Prism 6.0 (Graphpad). |
| Affinity data for this assay | |
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