Description
General description
All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction.
The ChIPAb+ LSD1 (KDM1A) set includes a LSD1 antibody, a Normal rabbit IgG and positive control primers which amplify a 85bp human prostate-specific antigen enhancer region. The LSD1 antibody and negative control IgG are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of LSD1 associated chromatin.
LSD1 (lysine specific demethylase 1), also known as BHC110, is a histone demethylase that specifically demethylates Lys4 of histone H3, a specific tag for epigenetic transcriptional activation, thereby acting as a corepressor. LSD1 acts by oxidizing the substrate by FAD to generate the corresponding imine that is subsequently hydrolyzed. LSD1 demethylates both mono- and tri-methylted Lys4 of histone H3, and may play a role in the repression of neuronal genes. Alone, it is unable to demethylate H3 Lys4 on nucleosomes and requires the presence of RCOR1/CoREST to achieve such activity. LSD1 may also demethylate Lys9 of histone H3, a specific tag for epigenetic transcriptional repression, thereby leading to derepression of androgen receptor target genes.
~110 kDa observed. Uniprot describes two isoforms at ~92 kDa and ~95 kDa due to alternative splicing
Immunogen
Histidine-tagged recombinant protein corresponding to full length LSD1.
Application
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from hormone starved (72 hrs), then androgen agonist induced (1 µM CI-4AS-1 overnight) LNCaP cells (~1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either Normal Rabbit IgG (Part No. PP64B), or 2 µg Anti-LSD1 (Part No.CS207350) and the Magna ChIP HiSens (Cat. # 17-10460). Successful immunoprecipitation of LSD1 associated DNA fragments was verified by qPCR using ChIP Primers, PSA (Part No. CS207340) as a positive locus, and ChIP Primers, β-globin (Part No. CS200591) as a negative locus.
Please refer to the Magna ChIP HiSens (Cat. # 17-10460) or EZ-Magna ChIP HiSens(Cat. # 17-10461) protocol for experimental details.
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from hormone starved (72 hrs), then androgen agonist induced (1 µM CI-4AS-1 overnight) LNCaP cells (1e5 and 1e6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either Normal Rabbit IgG (Part No. PP64B), or 2 µg Anti-LSD1 (Part No. CS207350) and the Magna ChIP HiSens Kit (Cat. # 17-10460). Successful immunoprecipitation of LSD1 associated DNA fragments was verified by qPCR using ChIP Primers, PSA (Part No. CS207340).
Please refer to the Magna ChIP HiSens (Cat. # 17-10460) or EZ-MagnaChIP HiSens (Cat. # 17-10461) protocol for experimental details
Western Blotting Analysis:
HeLa nuclear extract was probed with Anti-LSD1 (0.05 μg/mL).
Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Histones
Use ChIPAb+ LSD1 (KDM1A) ChIP Validated mouse monoclonal antibody for the detection of LSD1 (Lysine-specific histone demethylase 1A) also known as BRAF35-HDAC complex protein BHC110.
Packaging
25 assays per set. Recommended use: 2 μg of antibody per chromatin immunoprecipitation (dependent upon biological context).
Physical form
Affinity purified
Anti-LSD1 (Rabbit Polyclonal), Part No. CS207350. One vial containing 50 µg affinity purified rabbit polyclonal in 72 µL buffer containing 0.1M Tris-Glycine (pH 7.4), 150 mM NaCl and 0.05% sodium azide, before the addition of glycerol to 30%. Final concentration of antibody 0.7 mg/mL. Store at -20°C.
Normal rabbit IgG, Part No. PP64B. One vial containing 125 µg Rabbit IgG in 125 µL storage buffer containing 0.05% sodium azide. Store at -20°C.
ChIP Primers, PSA. Part No. CS207340. One vial containing 75 μL of 5 μM of each primer specific for human prostate-specific antigen (PSA) enhancer region (chr19:51354179-51354263, hg19 build). Store at -20°C.
FOR: GCC TGG ATC TGA GAG AGA TAT CAT C
REV: ACA CCT TTT TTT TTC TGG ATT GTT G
Preparation Note
Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.
Analysis Note
Chromatin Immunoprecipitation: Sonicated chromatin prepared from hormone starved (72 hrs), then androgen agonist induced (1 µM CI-4AS-1 overnight) LNCaP cells (~1e5 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either Normal Rabbit IgG (Part No. PP64B), or 2 µg Anti-LSD1 (Part No.CS207350) and the Magna ChIP® HiSens Chromatin IP Kit (Cat. # 17-10460). Successful immunoprecipitation of LSD1 associated DNA fragments was verified by qPCR using ChIP Primers, PSA (Part No. CS207340) (Figure 1).
Please refer to the MagnaChIP HiSens (Cat. # 17-10460) or EZ-MagnaChIP HiSens (Cat. # 17-10461) protocol for experimental details.
Control
Includes normal rabbit IgG and primers specific for a human prostate-specific antigen (PSA) enhancer region.
Other Notes
Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.
Legal Information
MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany
UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
