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LSD1 demethylation assay

WT Wen Juan Tu
RM Robert D. McCuaig
MM Michelle Melino
DR Daniel J. Rawle
TL Thuy T. Le
KY Kexin Yan
AS Andreas Suhrbier
RJ Rebecca L. Johnston
LK Lambros T. Koufariotis
NW Nicola Waddell
EC Emily M. Cross
ST Sofiya Tsimbalyuk
AB Amanda Bain
EA Elizabeth Ahern
NC Natasha Collinson
SP Simon Phipps
JF Jade K. Forwood
NS Nabila Seddiki
SR Sudha Rao
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LSD1 demethylation activity on custom made ACE2me2 peptides (Mimotopes) was assessed using the LSD1 fluorimetric drug discovery kit according to the manufacturer’s instructions (BML-AK544, Enzo Life Sciences). Briefly, ACE2me2 peptides were incubated with human recombinant LSD1 enzyme in LSD1/HRP assay buffer containing 1× HRP and 1× CELLestial® Red Peroxidase substrate. For LSD1 inhibition, various LSD1 inhibitors were incubated with recombinant LSD1 enzyme for 30 min at room temperature prior to adding ACE2me2 peptide. The fluorescent signal was measured in kinetic mode (40 s intervals) at an excitation of 530–570 nm, emission of 590 nm for 5–30 min using the Synergy H4 multi-mode plate reader system (BioTek).

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