Chromatin immunoprecipitation. ab500 ChIP Kit (abcam) was used according to the manufacturer’s instructions. Briefly, VSMCs were trypsinsed, centrifuged and then washed in ice-cold PBS. Cells were centrifuged again and then fixed in 1.1% Formaldehyde in PBS. Reactions were quenched with glycine and cells were washed in ice cold PBS before being lysed. Chromatin was sheared to approximately 500-1000 bp fragments using a sonicator at 4 °C. Chromatin was diluted and input chromatin was collected. Remaining chromatin was used for ChIP using 4 ug Runx2 (D130-3) as antibody of interest, 4 ug anti-histone H3 as a positive control and no antibody as a negative control. Antibodies were added for 12 hours at 4 °C and then Protein A sepharose beads were used to precipitate protein/DNA complexes. Cross-links were reversed by heating at 98 °C followed by Proteinase K addition and DNA purification. Samples were analysed by pPCR.
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提交于 Jun 04 2018