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Radioactive in vitro histone methyltransferase assay

DH Dominik Hoelper
HH Hongda Huang
AJ Aayushi Y. Jain
DP Dinshaw J. Patel
PL Peter W. Lewis
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A total of 1.0 µg of C-terminally K-biotinylated H3aa1–20 peptide (Tufts University Peptide Core Facility) were incubated with 20 µl of FLAG-hDAXX Mono Q fractions for 1 h at 30 °C in a reaction buffer containing 50 mM HEPES-KOH pH 7.9, 0.5 mM DTT, 2 mM MgCl2, and 0.25 mM PMSF. A total of 2 µl of radioactive [3H]-labeled S-adenosyl methionine was present during the reaction. For quantification, reactions were spotted onto P81 nitrocellulose filters (Whatman), filters were washed for three times using 100 mM sodium bicarbonate, dipped into acetone, and air-dried. Samples were subsequently subjected to liquid scintillation counting for activity measurement.

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