Doxycycline-Induced Biotinylated DNMT3A1 Immunoprecipitation

After a 16 h doxycycline (2 μg/mL) induction, whole-cell extracts were obtained from 5 × 10⁶ Bio-Dnmt3a1 ES cells by resuspension in CytoBuster Protein Extraction Reagent (Millipore) followed by 15-min incubation at room temperature (RT). Non-induced ESCs served as a negative control. Samples were spun at 20,000 g, 4 °C for 15 min and supernatant was isolated. Immunoprecipitation of biotinylated-DNMT3A1 was performed with M280-streptavidin beads (Thermo Fisher) using whole-cell extract from cultures with or without doxycycline induction. Equal amounts of protein were resuspended in Laemmli buffer and loaded for western blot analysis. The following antibodies were used: anti-DNMT3A (Santa Cruz, sc-20,703), anti-TET1 (provided by Dr. Guoliang Xu, [24 (link)]), and anti-β-actin (Santa Cruz, sc-47,778).

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Gu T., Lin X., Cullen S.M., Luo M., Jeong M., Estecio M., Shen J., Hardikar S., Sun D., Su J., Rux D., Guzman A., Lee M., Qi L.S., Chen J.J., Kyba M., Huang Y., Chen T., Li W, & Goodell M.A. (2018). DNMT3A and TET1 cooperate to regulate promoter epigenetic landscapes in mouse embryonic stem cells. Genome Biology, 19, 88.

Publication 2018

CytoBuster Protein Extraction Reagent M280-streptavidin beads anti-DNMT3A anti-TET1 anti-β-actin

Actin Antibodies Cell extract Cells Doxycycline Escs Immunoprecipitation Laemmli buffer Protein Streptavidin Western blot analysis

Corresponding Organization :

Other organizations : Baylor College of Medicine, The University of Texas MD Anderson Cancer Center, Minneapolis Heart Institute Foundation, University of Minnesota, Prevention Institute, Texas A&M University, Stanford University, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences

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Variable analysis

independent variables

Doxycycline induction (2 μg/mL)

dependent variables

Expression of DNMT3A1 protein
Interaction between DNMT3A1 and TET1 proteins

control variables

Cell line (Bio-Dnmt3a1 ES cells)
Incubation time (16 h)
Cell number (5 × 10^6)
Protein extraction method (CytoBuster Protein Extraction Reagent)
Incubation time for protein extraction (15 min)
Centrifugation conditions (20,000 g, 4 °C, 15 min)

positive control

Whole-cell extract from cultures with doxycycline induction

negative control

Non-induced ESCs

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