ChIP Assay of Chromatin Modifications

For ChIP assays, K562 cells were fixed with 1% (vol/vol) formaldehyde for 10 min at room temperature. ChIP assays were performed using the EZ-Magna ChIP A/G Kit (Millipore). Anti-Bmi1 antibody (Active Motif, AF27), Anti-Ring1b antibody (MBL International, D139-3), Anti-H3K9Ac antibody (Millipore, 07-942), Anti-H3K4me3 antibody (Millipore, 07-473), anti-H3K27me3 antibody (Millipore, 07-449), Anti-H2AK119ub1 antibody (Cell Signaling, D27C4) and normal mouse IgG were used for immunoprecipitation. ChIP DNA was then subjected to real-time PCR analysis using primers targeting different region of ribosomal protein gene promoters [23 (link)-24 (link)].

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Gao R., Chen S., Kobayashi M., Yu H., Zhang Y., Wan Y., Young S.K., Soltis A., Yu M., Vemula S., Fraenkel E., Cantor A., Antipin Y., Xu Y., Yoder M.C., Wek R.C., Ellis S.R., Kapur R., Zhu X, & Liu Y. (2015). Bmi1 promotes erythroid development through regulating ribosome biogenesis. Stem cells (Dayton, Ohio), 33(3), 925-938.

Publication 2015

EZ-Magna ChIP A/G Kit Anti-H3K9Ac antibody Anti-H3K4me3 antibody anti-H3K27me3 antibody

Anti antibody Bmi1 Chip Chip assays Formaldehyde Gene promoters region H3k4me3 Immunoprecipitation K562 cells Mouse Primers Real time pcr analysis Ribosomal protein Ring1b

Corresponding Organization : Indiana University – Purdue University Indianapolis

Other organizations : Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Massachusetts Institute of Technology, Dana-Farber Cancer Institute, Harvard University, Memorial Sloan Kettering Cancer Center, University of California, San Diego, University of Louisville

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Variable analysis

independent variables

Antibody used for immunoprecipitation (Anti-Bmi1, Anti-Ring1b, Anti-H3K9Ac, Anti-H3K4me3, Anti-H3K27me3, Anti-H2AK119ub1)

dependent variables

Binding of antibodies to different regions of ribosomal protein gene promoters (measured by real-time PCR analysis)

control variables

K562 cell line
Formaldehyde concentration (1% vol/vol)
Formaldehyde fixation time (10 min)
Formaldehyde fixation temperature (room temperature)
ChIP assay kit (EZ-Magna ChIP A/G Kit)
Normal mouse IgG as negative control

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