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+JMJD1C set includes the JMJD1C antibody, a Normal Rabbit IgG, and control primers which amplify a 110 bp region of ChIP Primers, human β-globin. The JMJD1C and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of JMJD1C -associated chromatin.
JmjC domain-containing protein 6 (JMJD6) is a dioxygenase belonging to group of proteins known as the Jmjc-domain containing proteins. This family of proteins is a group of redox enzymes specifically involved in catalyzing oxidation reactions, and are characterized by the presence of a highly conserved jumonji C (Jmjc)-domain. JMJD6 can function both as a lysyl-hydroxylase and a histone arginine demethylase. As a lysyl-hydroxylase, JMJD6 catalyzes 5-hydroxylation of target protein lysine residues. As an arginine demethylase, JMJD6 has a critical part to play in histone code by demethylating of histone H3 and histone H4. It has also been shown to mediate U2AF2/U2AF65 5-hydrozylation, thereby functioning as a regulator for RNA splicing through affecting U2AF2/U2AF65 pre-mRNA activity. JMJD6 is a critical component of embryogenesis for the differentiation of various organs, and may act as a regulator for hematopoietic regulation and macrophage cytokine response. High expression of JMJD6 has been observed in heart, kidney and skeletal muscle. Medium to low expression has been seen in brain, liver, lung, ovary, pancreas, placenta, prostate, testis, thymus, and spleen.
~46 kDa observed. Uncharacterized crossreacting band observed below the 46 kDa target band in some lysates.
Immunogen
Linear peptide corresponding to human JMJD6.
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 1.4 µg of either Normal Rabbit IgGor Anti-JMJD6 and the Magna ChIP A Kit (Cat. # 17-610). Successful immunoprecipitation of JMJD6 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 resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-JMJD6 (1 µg/mL). Proteins were visualized using a Donkey Anti-Rabbit IgG conjugated to HRP and chemiluminescence detection system.
Arrow indicates JMJD6 (~46 kDa). (Figure 3).
Immunocytochemistry Analysis:
Representative lot data.
Confocal fluorescent analysis of A431, HeLa cells using Anti-JMJD6 (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
Histone Modifying Proteins
This ChIPAb+ JMJD6 -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Biochem/physiol Actions
This antibody recognizes JMJD6.
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
Anti-JMJD6 (rabbit polyclonal). One vial containing 50 µL of purified rabbit polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4), 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 1.4 µg of either Normal Rabbit IgG or Anti-JMJD6 and the Magna ChIP® A Kit (Cat. # 17-610). Successful immunoprecipitation of JMJD6 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.
