| Assay Method Information | |
| | LSD1 in vitro activity assay |
| Description: | Principle: LSD1 specifically removes the methylation modification at K4 lysine on H3 polypeptide substrate, making it a substrate without methylation. The method employed histone H3 methylated polypeptide (1-24) as a substrate and introduces a biotin label in the C segment of the substrate. When LSD1 was initiated with the participation of FAD, the methylation modification on the substrate H3K4 can be removed. The Eu-labeled H3K4 background antibody binded to the substrate by antigen-antibody reaction, while the streptavidin-labeled receptor binded together by the specific interaction of streptavidin and biotin, thereby the Eu-labeled donor interacting with the streptavidin-labeled receptor. In fluorescence resonance energy transfer, when two fluorophores were brought close due to biomolecular interaction, part of the energy captured by the cryptate when excited would be released, the emission wavelength of which is 620 nm; the other part of the energy was transferred to the acceptor, the emission wavelength of which is 665 nm. The 665 nm emission can only produce by FRET caused by the donor. Therefore, when biomolecules interact, there were two excited lights at 620 nm and 665 nm; when there was no interaction, there was only one excited light at 620 nm. The LSD1 demethylation activity can be reflected by detecting the ratio between the fluorescence signals at the two emission wavelengths of 665 nm and 620 nm. Meanwhile, a blank control was set to determine the strength of the enzyme activity. |
| Affinity data for this assay | |
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