Assay Method Information

Assay Name:  AlphaScreen Assay
Description:  Demethylase reactions and AlphaScreen assays were performed as described (Sayegh et al., 2013), with a few exceptions. All demethylase buffers contained 125 μM αKG, 50 μM (NH4)2Fe(SO4)2 and 64 nM C-terminal H3K4me3(1-20)-GGK-biotinylated peptides (Sayegh et al., 2013). The enzymes were used at the following concentrations: 19 nM FLAG-KDM5A, 25 nM FLAG-KDM5B, 8 nM FLAGKDM5C, 29 nM His-FLAG-KDM6A, and 8 nM FLAG-KDM6B. Demethylation reactions contained 0.05% DMSO. The AlphaScreen general IgG (protein A) detection kit from PerkinElmer Life Sciences was used as described (Sayegh et al., 2013). The AlphaScreen optic module on a Pherastar (BMG Labtech) microplate reader was used to record luminescence emission at 570 nm.
Affinity data for this assay
 

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