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+ CTCF set includes the CTCF antibody, a negative control antibody (normal mouse IgG), and qPCR primers which amplify a 165 bp region of human H19 internal control region. The CTCF and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of CTCF-associated chromatin.
CTCF, also known as CCCTC-binding factor, contains 11 zinc finger (ZF) binding domains that allow it to bind different DNA target sequences and proteins. This multifunctional protein can act as a transcriptional activator or repressor. As a transcriptional repressor, CTCF, binds to promoters of vertebrate c-myc gene. It also binds to the PLK and PIM1 promoters. CTCF may prevent the access of transcriptional activators to enhancers. CTCF also acts as a transcriptional activator of APP. It is involved in different aspects of gene regulation including promoter activation or repression, hormone-responsive gene silencing, methylation-dependent chromatin insulation, and genomic imprinting. Mutations of CTCF in this gene have been associated with invasive breast cancers, prostate cancers, and Wilms tumors, suggesting a role for CTCF may as a tumor suppressor.
~ 130 kDa
Note: Poly(ADP-ribosyl)ated isoform, C-terminal truncation, or sumoylation will cause variable migration of CTCF protein on SDS–PAGE, which may present CTCF with different molecular weight on Western Blot. Modifications are dependent on cell type and growing condition.
The following molecular weights have been reported in the literature: 180 kDa (CTCF-180), 130 kDa (CTCF-130), 82 kDa (CTCF-82), 70 kDa (CTCF-70). See Klenova,E.M.,et al, (1997) Nucleic Acids Res. 25, 466–473 and Torrano,V,et al, (2006) Journal of Cell Science 119, 1746-1759) for additional details.
Immunogen
His-tagged recombinant protein corresponding to human CTCF.
Application
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP with 15 min fixation) were subjected to chromatin immunoprecipitation using 2 µg of either Normal Mouse IgG or 2 µg of Anti-CTCF and the Magna ChIP G Kit (Cat. # 17-611).
Successful immunoprecipitation of CTCF associated DNA fragments was verified by qPCR using ChIP Primers, Human H19 ICR as a positive locus, and a representative published negative locus (Wendt, K.S., et al., 2008) (Please see figures). 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.
K562 cell lysate was resolved by electrophoresis, transferred to PVDF membranes and probed with Anti-CTCF, clone 1.2.1.5.4 (1 μg/mL).
Proteins were visualized using a Goat Anti-Mouse conjugated to HRP and a chemiluminescence detection system (Please see figures).
The following molecular weights have been reported in the literature: 180 kDa (CTCF-180), 130 kDa (CTCF-130), 82 kDa (CTCF-82), 70 kDa (CTCF-70). See Klenova, E.M., et al, (1997) Nucleic Acids Res. 25, 466–473 and Torrano,V., et al, (2006) Journal of Cell Science 119, 1746-1759) for additional details.
Please see molecular weight section for additional information.
The ChIPAb+ CTCF set includes the CTCF antibody, a negative control antibody (normal mouse IgG) & qPCR primers which amplify a 165 bp region of human H19 internal control region.
Biochem/physiol Actions
Recognizes CTCF, Mr ~130 kDa.
Physical form
Anti-CTCF, (mouse monoclonal IgG1κ. One vial containing 50 μg of protein G purified mouse monoclonal IgG1κ in buffer containing 0.1 M Tris-Glycine (pH 7.4, 150 mM NaCl) with 0.05% sodium azide, before the addition of glycerol to 30%. Store at -20°C.
Normal Mouse IgG. Two vials containing 25 µg purified IgG in 25 µL storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers, Human H19 ICR. One vial containing 75 μL of 5 μM of each primer specific for human H19 internal control region. Store at -20°C.
FOR: CCC ATC TTG CT GACC TCAC
REV: AGA CCT GGG ACG TTT CTG TG
Format: Purified
Analysis Note
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10⁶ cell equivalents per IP with 15 min fixation) were subjected to chromatin immunoprecipitation using 2 µg of either Normal Mouse IgG or 2 µg of Anti-CTCF and the Magna ChIP® G Kit (Cat. # 17-611). Successful immunoprecipitation of CTCF associated DNA fragments was verified by qPCR using ChIP Primers Human H19 ICR (Please see figures).
Please refer to the EZ-Magna ChIP A (Cat. # 17-408) or EZ-ChIP (Cat. # 17-371) protocol for experimental details.
Control
Includes negative control mouse IgG antibody and primers specific for human H19 ICR.
Legal Information
MAGNA CHIP is a registered trademark of Merck KGaA, Darmstadt, Germany
UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany
