Epiquik dna methyltransferase activity inhibition assay kit

DNA methyltransferase (DNMT) activity in nuclear extracts prepared from control and treated prostate stem cells and prostate tissues was determined by EpiQuik™ DNA Methyltransferase Activity/Inhibition Assay Kit (EpiGentek, Farmingdale, NY). In this assay, a unique cytosine-rich DNA substrate is stably coated on the test strip wells. DNMT enzymes in the samples transfer a methyl group to cytosine from Adomet (S-adenosylmethionine) to methylate the DNA substrate. The methylated DNA is recognized by an anti-5-methylcytosine antibody. The methylated DNA level was proportional to the enzyme activity, and was detected by a detection antibody, with enhancer and developer solutions. The developed color was quantified at 450 nm in a plate reader (FLUOstar).

The inhibitory activity of the 5-aza treatment was validated by the estimation of DNMT activity in the treated insects. For this, the nuclear protein was extracted from the treated and untreated (control) insects using EpiQuik Nuclear Extraction Kit (Epigentek, Farmingdale, NY, USA), following the manufacturer’s instructions. The extracted protein was quantified using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Next, DNMT activity was estimated using 3 μg of the nuclear protein extract for each sample (i.e., treated and untreated) in triplicates, using the EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek, Farmingdale, NY, USA), as per the manufacturer’s protocol. Pure DNA methyltransferase supplied with the kit was used as a positive control. Methyltransferase activity was calculated using the average absorbance at 450 nm and was expressed as OD/hr/mg. Subsequently, DNMT activity in the treated insects was compared with that of the untreated (control) insects, and a percent reduction in DNMT levels was estimated.

The nuclear protein at 10 μg extracted from the Bm12 cells was treated with 5-aza-dC or PBS (as control). DNA methyltransferase activity was analyzed using the EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek) following the manufacturer’s instructions. Pure mouse methyltransferase DNMT1 in the kit was used as a positive control. Methyltransferase activity is presented as the average absorbance at 450 nm.

Protein was isolated from treated cells (as previously described) using EpiQuik Nuclear Extraction Kit II (Epigentek, Farmingdale, NY, USA) following the manufacturer's directions. DNMT inhibition was analyzed using EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (P-3001-2, Epigentek) following the manufacturer's directions.

Nuclear extracts of BMDM were prepared using the EpiQuik Nuclear Extraction Kit (OP-0002-1, Epigentek), following their protocol, generating nuclear and cytoplasmic extracts. The protein concentration of the nuclear extract was determined using the bicinchoninic acid protein assay (PI23235, ThermoFisher Pierce), following the manufacturer's protocol. To determine DNMT activity, we used the EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (P-3001-2, Epigentek) and followed the manufacturer's protocol. BMDM nuclear extracts were used in this assay. To determine TET activity, we used the Epigenase 5mC-Hydroxylase TET Activity/Inhibition Assay Kit (P-3086-96, Epigentek) and followed the manufacturer's protocol. BMDM nuclear extracts were used in this assay.

DNA methyltransferase (Dnmt) activity was measured by EpiQuik DNA Methyltransferase Activity/Inhibition Assay Kit (Epigentek, Brooklyn, NY, USA) using nuclear extracts from undifferentiated (UC), osteo/odontogenic cells (OB) and osteo/odontogenic cells plus procaine (OB+Procaine). Nuclear extracts were isolated with the EpiQuik Nuclear Extraction Kit (Epigentek). Dnmt activity assay was performed according to the manufacturer’s instructions. After incubation, capturing, and developing enzyme activity for samples and controls, absorbance was measured on a microplate reader at 450 nm and Dnmt activity (optical density [OD]/mg/h) was calculated according to the following formula: [(sample OD—blank OD)/(sample protein x incubation time)] x 1000.