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| | In vitro HDACs Inhibition Fluorescence Assay |
| Description: | Bos-Lys (acetyl)-4-amino-7-methylcoumarin substrate was used in inhibition assays against class I (HDAC1, HDAC2, HDAC3) and class IIb (HDAC6), while Boc-Lys (trifluoroacetyl)-4-amino-7-methylcoumarin substrate for class IIa (MDA-MB-231 cell lysate). 10 µL of enzyme solution was mixed with various concentrations of tested compounds (50 µL). Five minutes later, 40 µL of fluorogenic substrate was added. The mixture was incubated at 37°C for 30 minutes, stopped by addition of 100 µL of developer containing trypsin and TSA, and then incubated again at 37°C for 20 min. Fluorescence intensity was measure using a microplate reader at excitation and emission wavelengths of 390 and 460 nm, respectively. |
| Affinity data for this assay | |
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