Total protein extract from cells was prepared using RIPA buffer (Sigma) and used for in vitro DNA methyltransferase-1 protein quantification by EpiQuik Dnmt1 Assay Kit (Epigentek, P-3011-3). Initially the standard curve was generated with purified DNMT1 protein (provided by manufacturer) and the protein amount was quantified from cells extracts (Supplementary Figure S10 ). Experiments were done in biological triplicates and statistical analysis was performed with independent samples t-test using Graph Pad Software.
Epiquik dnmt1 assay kit
Manufactured by Epigentek
Sourced in United States
The EpiQuik Dnmt1 Assay Kit is a laboratory tool designed to measure the DNA methyltransferase 1 (Dnmt1) activity in various sample types. It provides a quantitative assessment of Dnmt1 levels without making claims about its intended use or interpretation of the results.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (1 mentions)
Microplate spectrophotometer, by Agilent Technologies (1 mentions)
Ab13537, by Abcam (1 mentions)
Epiquik dna methyltransferase activity inhibition assay kit, by Epigentek (1 mentions)
5 protocols using epiquik dnmt1 assay kit
1
Quantifying Cellular DNMT1 Protein
2
Quantification of DNMT1 Levels in Lens Capsule
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Nuclear proteins were isolated from lens capsule samples using the same method as described in Section 2.9 . To measure the amount of DNMT1 protein in samples, the EpiQuik™ DNMT1 assay kit (#P-3011, Epigentek Group, USA) was used according to the manufacturer's protocol. Briefly, the samples and the DNMT1 antibody were incubated for 120 min at 37 °C. Subsequently, they were incubated with developing solution for 10 min at room temperature. After the reactions were stopped by addition of the stopping solution, the absorbance was measured at 450 nm using a microplate spectrophotometer (Biotek, USA).
3
Investigating Oct4 and Dnmt1 Expression
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Real-time PCR and western blotting were conducted to investigate the mRNA and protein expression levels of mouse Oct4 and confirm whether Dnmt1 was downregulated. Total RNA extraction, cDNA synthesis, and real-time PCR were performed as described above. Total proteins were extracted from the RNAi-transfected CCE cells following the procedure detailed in the manual of the “NE-PER Nuclear and Cytoplasmic Extraction Reagents” (#78833, Thermo Fisher Scientific). The protein concentration was measured using a bicinchoninic acid assay on a NanoDrop 2000 spectrophotometer. Western blotting analysis was performed as described previously [37 (link)]. The antibody against mouse Dnmt1 was purchased from Abcam (ab13537). For the Dnmt1 assay, the RNAi-transfected CCE cells were initially lysed with NE-PER Nuclear and Cytoplasmic Extraction Reagent, and the EpiQuik DNMT1 assay kit (Epigentek, P-3011) was used to detect the amount of Dnmt1 according to the manufacturer’s instructions and as described previously [38 (link)].
4
Quantifying DNMT-1 Protein in Nuclear Extracts
5
Quantification of DNMT1 and Activity
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The nuclear extracts were obtained as previously described [16 (link)]. DNMT1 (DNA methyltransferase) quantification and DNMT activity assays were performed using 5 μg of nuclear extract, an EpiQuik™ DNMT1 Assay Kit and an EpiQuik™ DNA Methyltransferase Activity/Inhibition Assay Kit (EpiGentek), respectively, according to the manufacturer’s instruction.
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