Epiquick nuclear extraction kit

From the frozen tissue prepared as described above, nuclear extracts were prepared using the Epiquick Nuclear Extraction Kit (Epigentek) according to manufacturer instructions. Protein concentrations were determined using a Bradford Assay kit (Pierce) according to kit directions for 96-well plates. To measure histone deacetylase activity, the EpiQuik HDAC Activity/Inhibition Assay Kit (Epigentek) was used, and to measure DNA methyltransferase activity, the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (Epigentek) was used, both per the manufacturer’s instructions. All samples and controls were run in duplicate to reduce technical error. For VMN nuclear extract, 2.5μg of protein was loaded per well in the HDAC assay, and 5μg was loaded for the DNMT assay. For MePD nuclear extract, 2.5μg was loaded per well in the HDAC assay, and 3ug was loaded for the DNMT assay. Incubations were for 120min for DNMT assay and 90min for HDAC assay, as recommended in both cases. Samples were read after 8min of development on a Tecan Infinite M1000 Pro, and raw and reference ODs were measured. To calculate HDAC and DNMT activity (OD/h/mg), the following conversion equation, as recommended by the Epigentek Protocol, was used:
$$[(\text{Sample}\text{OD}-\text{Blank}\text{OD})/(\text{Protein}\text{amount}\mathrm{\mu }\mathrm{g}\ast \text{hours}\text{incubated})]\ast 1000=\text{Activity}(\text{OD}/\mathrm{h}/\text{mg})$$