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LSD1 (human), (recombinant)

Highly active
 
BML-SE544-0050 50 µg 400.00 USD
 

Product Specification

Alternative Name:KDM1, Lysine-specific histone demethylase 1, BHC110, AOF2, KIAA0601
 
Sequence:N-terminal truncation of LSD1 from human cDNA. Sequence is identical to Genbank accession NM015013 (aa 151-852).
 
MW:78 kDa
 
Source:Produced in E. coli.
 
UniProt ID:O60341
 
Formulation:Liquid. In 8.1mM Na2HPO4, 1.5mM KH2PO4, pH 7.2, 138mM sodium chloride, 2.7mM KCl and 40% v/v glycerol.
 
Purity:≥90% (SDS-PAGE)
 
Purity Detail:Purified by multi-step chromatography.
 
Activity:Highly active in a peroxidase-coupled assay with the Histone H3 Dimethyl Lysine-4 Peptide (Prod. No. BML-P256).
 
Specific Activity:≥20 U/µg. One U=1 pmol/min at 30°C, 100 µM Histone H3 Dimethyl Lysine-4 Peptide (Prod. No. BML-P256).
 
Application:Study LSD1 kinetics and inhibitor sensitivity and also the effect of the enzyme in transcriptional regulation, cell cycle progression, and oncogenesis.
 
Long Term Storage:-80°C
 
Use/Stability:The enzyme is stable on ice for the time typically required to set up an experiment (30-60 min.), but may lose activity with prolonged storage on ice. It is recommended that thawing and dilution of the enzyme be done within as short a time as possible before start of the assay. The remaining, unused, undiluted enzyme should be refrozen quickly by, for example, snap freezing in a dry/ice ethanol bath or liquid nitrogen. Freezing and storage of diluted enzyme is not recommended.
 
Handling:Do not freeze/thaw. After opening, prepare aliquots and store at -80°C.
 
Miscellaneous/General:The mono, di- and trimethylation of particular lysine residues in histone tails (e.g. histone H3 lysine-4 (H3K4), H3K9, H3K27, H3K36, H4K20) are implicated, along with a variety of other post-translational modifications (e.g. lysine acetylation) in the transmission of heritable epigenetic information and the control of chromatin structure and DNA transcription. LSD1 catalyzes the oxidative demethylation of mono- and dimethylated H3K4, producing hydrogen peroxide and formaldehyde in the process. H3K4 methylation is considered a transcription activating chromatin mark and in vivo LSD1 is frequently found in association with the transcriptional corepressor protein CoREST and HDACs 1 or 2. LSD1 is inhibited by a number of established monoamine oxidase inhibitor drugs. That and the fact that its expression is elevated in a number of cancers may make it a promising target for drug development.
 
Protocol:Assay of LSD1 (Catalog # BML-SE544) by a Peroxidase-Coupled Assay with Histone H3 Dimethyl Lysine-4 Peptide (H3 K4Me2), Cat. # BML-P256)
Components of the Assay:
LSD1 Assay Buffer (LAB): (25mM KH2PO4KOH, pH7.8, 5% v/v glycerol)
LSD1 (Cat. # BML-SE544): Dilute to enough LSD1 to 0.05μg/μl with LSD1 Assay Buffer to provide 10μl per well. Keep on ice until use.
4-Aminoantipyrine (4-AA): Prepare a 5mM stock in dH2O. Store at -20°C or short term at 4°C.
3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS): Prepare a 50mM stock in dH2O. Store at -20°C or short term at 4°C.
Horse Radish Peroxidase (HRP): Prepare a 100μM stock in dH2O and keep on ice. Prepare fresh daily and discard remainder.
Peroxidase Reaction Mix (PRM): The peroxide generated by the LSD1-catalyzed demethylation of H3 K4Me2 will produce, in an HRP-catalyzed reaction, a colored adduct of 4-AA and DCHBS with an extinction coefficient of 26,000 M-1cm-1 at 515nm. PRM is: 7.5μM HRP, 0.25mM 4-AA, 2.5mM DCHBS diluted in LAB. Plan to prepare enough PRM to provide 40μl per well. The 0.05μg/μl LSD1 dilution (10μl) will be added to the 40μl of PRM and later 50μl of a 2x substrate solution will be added (see below). If, for example, you plan to pre-incubate the LSD1 with a potential inhibitor prior to addition of the peptide substrate, it may be desirable to prepare aliquots of PRM with the inhibitor added to 2.5 times its final concentration.
Histone H3 Dimethyl Lysine-4 Peptide (H3 K4Me2) (Cat. # BML-P256): Prepare a 0.5mM stock of of H3 K4Me2 by dissolving 0.5mg net peptide (1 vial of Cat. # BML-P256) in 438μl of LSD1 Assay Buffer.
2x Substrate Solution: Prepare a 2x substrate solution by diluting the 0.5 mM H3 K4Me2. Each assay well will require 50 μl (see below). For example, prepare 1 ml of 20 μM substrate (for final 10 μM) by mixing 40μl of 0.5 mM H3 K4Me2 and 960μl LAB. (NOTE: LSD1’s Km for H3 K4Me2 is 9.2μM. Therefore, a convenient substrate concentration for inhibitor screening Might be 10μM H3 K4Me2, while 100μM would be more suitable for a specific activity measurement at a saturating substrate concentration.) Warm to 30°C before use. If, for example, you plan to expose LSD1 to a potential inhibitor simultaneously with the addition of the peptide substrate, it may be desirable to prepare aliquots of 2x Substrate Solution with the inhibitor added to 2.0 times its final concentration.
Clear-well ½-volume 96-well microplate
Reaction Condition Examples:
1) Designate wells for two reactions: one well for +LSD1 and one no-enzyme control.
2) Add 40μl of PRM to both wells. Allow the microplate to equilibrate to assay temperature (30°C).
3) Add 10μl of diluted LSD1 (BML-SE544, 0.05μg/μl) to the +LSD1 well and 10μl LAB to the no-enzyme well.
4) To start reactions, add 50μl of the 2x Substrate (30°C) to both wells.
5) Data analysis: Determine the rate of the enzyme reaction in mOD/min at 515nm, by, for example, determining the slope of the initial, linear part of a graph of absorbance (mOD units) versus time (min.) The extinction coefficient of the colored reaction product is 26,000 M-1cm-1 and the optical path-length of 100μl in a ½-volume 96-well microplate is ~0.5 cm. (NOTE: Some plate-reading spectrophotometers have a feature that allows the path-length to measured empirically for each well.) If a path-length of 0.50cm is assumed, rates in mOD515nm/min may converted to pmol/min with a conversion factor of 7.7pmol/mOD515nm.
 
BML-SE544 SDS-PAGE
SDS-PAGE Analysis: Lane 1: MW Marker, Lane 2: 0.5 µg of Purified LSD1 (human) (rec.) Prod. No. BML-SE544.
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BML-SE544 SDS-PAGE

General Literature References

LSD1: oxidative chemistry for multifaceted functions in chromatin regulation.: F. Forneris, et al.; Trends Biochem. Sci. 33, 181 (2008), Abstract;
Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications.: M. Lee, et al.; Chem. Biol. 13, 563 (2006), Abstract;
Histone demethylation catalysed by LSD1 is a flavin-dependent oxidative process.: F. Forneris, et al.; FEBS Lett. 579, 2203 (2005), Abstract;
Human histone demethylase LSD1 reads the histone code.: F. Forneris, et al.; J. Biol. Chem. 280, 41360 (2005), Abstract;
Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.: Y. Shi, et al.; Cell 119, 941 (2004), Abstract;

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