Analysis Note

ControlIncludes normal rabbit IgG and primers specific for human p21.

Application

This ChIPAb+ N-CoR -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Research Sub CategoryHormones & Receptors

Research CategoryEpigenetics & Nuclear Function

Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from untreated or UV treated (6 hrs, 50 joules/m2.) U2OS cells (3 X 10E6 cell equivalents per IP) was subjected to chromatin immunoprecipitation using using 3 µg of either Normal Rabbit IgGor 3 µg of Anti-N-CoR and the Magna ChIP™ A/G Kit (Cat. # 17-10085). Successful immunoprecipitation of N-CoR associated DNA fragments was verified by qPCR using ChIP Primers, p21. (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.
RAW 264.7 cell lysate was probed with Anti-N-CoR (1 µg/mL). Proteins were visualized using a Donkey Anti-Rabbit IgG secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates Anti-N-CoR (~270 kDa). (Figure 3).

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.

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.

In the absence of ligands, the corepressor N-CoR represses nuclear hormone receptor-mediated gene transcription. In the presence of ligands, these hormone receptors activate gene expression and play critical roles in development, growth, and homeostasis. N-Cor-mediated transcriptional repression is facilitated through the formation of histone deacetylase (HDACs) N-CoR complexes or by conjugation from small ubiquitin-like modifier 1 (SUMO-1). It is shown that the SANT1 domain of N-CoR serves as a binding region for SUMO-E2 conjugating enzyme Ubc9 and SUMO-E3 ligase Pias1, which is necessary for N-CoR-mediated repression of respective reporter genes. SUMOylation-deficient N-COR knockout mice display a signification reduction in the repression of gene transcription and result in defects involving the differentiation and development of neural cells into erythrocytes and thymocytes.

Immunogen

KLH-conjugated linear peptide corresponding to a region between amino acids 1510 and 1540 of the N-CoR protein.

Legal Information

MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Packaging

25 assays per set. Recommended use: 3 µg of antibody per chromatin immuno-precipitation (dependent upon biological context).

Physical form

Affinity purified

Components:
Anti-N-CoR (Rabbit Polyclonal). One vial containing 75 µg of affinity purified polyclonal in buffer containing 0.1M Tris-glycine (pH 7.4) and 150 mM NaCl with 0.05% sodium azide before the addition of 30% glycerol. 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, p21. One vial containing 75 µL of each primer (5 µM) specific for the human p21 (WAF1/CIP1/CDKN1A) promoter. Store at
-20°C.
FOR: CCC ACA GCA GAG GAG AAA GAA
REV: CTG GAA ATC TCT GCC CAG ACA

Quality

Chromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from U2OS cells (3 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 3 µg of either Normal Rabbit IgGor 3 µg of Anti-N-CoR and the Magna ChIP^® A/G Kit (Cat. # 17-10085). Successful immunoprecipitation of N-CoR associated DNA fragments was verified by qPCR using ChIP Primers, p21flanking an Sp1 binding site in the human p21 promoter. (Figure 1).
Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.

Storage and Stability

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.

Target description

~270 kDa observed. Uniprot describes 2 isoforms produced by alternative splicing at MW of 266 kDa and 270 kDa