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+JHDM1B set includes the JHDM1B antibody, a Normal Rabbit IgG, and control primers which amplify a 110 bp region of ChIP Primers, human β-globin. The JHDM1B and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of JHDM1B -associated chromatin.
JHDM1B (jumonji domain-containing histone demethylation protein 1B) and KDM2B. Due to the presence of a F-box motif, this protein is also known as the F-box/LRR-repeat protein 10 (FBXL 10). This protein functions as a histone demethylase for lysine 36 of histione H3 and preferentially demethylates dimethylated lysine 3 with weak or no activity for mono- and tri-methylted H3 lysine 36.
~165 kDa observed. An uncharacterized lower molecular weight band may be observed in some lysates.
Immunogen
KLH-conjugated linear peptide corresponding to human JHDM1B.
Application
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µL of either Normal Rabbit IgG or Anti-JHDM1B and the Magna ChIP A Kit (Cat. # 17-610). Successful immunoprecipitation of JHDM1B associated DNA fragments was verified by qPCR using ChIP Primers, human β-globin as a positive locus, and β-actin promoter primers as a negative locus. (Figure 2). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Western Blot Analysis:
Representative lot data.
HeLa nuclear extract was probed with Anti-JHDM1B (1 ug/mL). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates JHDM1B (~165 kDa). (Figure 3). An uncharacterized band appears at ~109 kDa in some lysates.
Immunocytochemistry Analysis:
Representative lot data.
Confocal fluorescent analysis of NIH/3T3 and HeLa cells using Anti-JHDM1B (Red). Actin filaments have been labeled with Alexa Fluor 488 dye -Phalloidin (Green). Nucleus is stained with DAPI (Blue). This antibody positively stains the nucleus. (Figure 4).
Research Category
Epigenetics & Nuclear Function
Research Sub Category
Histones
This ChIPAb+ JHDM1B -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Packaging
25 assays per set. Recommended use: 1.4 μg (2 μL) of antibody per chromatin immuno-precipitation (dependent upon biological context).
Physical form
Affinity purified
Components:
Anti-JHDM1B (Rabbit Polyclonal). One vial containing 50 µL of purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4) and 150 mM NaCl and 0.05% sodium azide before the addition of glycerol to 30%. Store at -20°C.
Concentration: 0.7 mg/mL
Normal Rabbit IgG. One vial containing 125 µg of Rabbit IgG in 125 µL of storage buffer containing 0.05% sodium azide. Store at -20°C.
ChIP Primers, human β-globin. One vial containing 75 μL of each primer (5 μM) specific for the human β-globin promoter. Store at -20°C.
FOR: AGG ACA GGT ACG GCT GTC ATC
REV: TTT ATG CCC AGC CCT GGC TC
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.
Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.
Analysis Note
Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µL of either Normal Rabbit IgG or Anti-JHDM1B and the Magna ChIP® A Kit (Cat. # 17-610). Successful immunoprecipitation of JHDM1B associated DNA fragments was verified by qPCR using ChIP Primers, human β-globin (Figure 1).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Control
Includes normal rabbit IgG and primers specific for human β-globin.
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.
