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| | HDAC Cell-Free Assays |
| Description: | Briefly, recombinant HDACs and substrates were diluted in reaction buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7mM KCl, 1mM MgCl2, 1 mg/ml BSA, 1% dimethylsulfoxide). 5 nl of serial diluted CQ (in dimethyl sulfoxide) was added with the Echo 550 liquid handler (Labcyte Inc). According to the enzymatic specificity, a fluorogenic peptide from p53 residues 379-382 (RHKK(Ac)) was used as the substrate for Class I (HDAC1, -2, -3, and -5), Class IIb (HDAC6 and -10), and class IV HDACs (HDAC11), and a fluorogenic peptide from p53 residues 379-382 (RHK(Ac)K(Ac)) was used for HDAC8, whereas Boc-Lys(trifluoroacetyl)-AMC was used for Class IIA HDACs (HDAC4, -5, -7, and -9). The reaction was started by adding substrates into the reaction mixture and was stopped after 2 h of incubation at 30 °C. Substrate concentrations were analyzed at excitation and emission wavelengths of 360 and 460nm. |
| Affinity data for this assay | |
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