ENCAB247TYO
Antibody against Homo sapiens CTCF, Mus musculus CTCF
Homo sapiens
K562, GM12878, HepG2, MCF-7, C4-2B, HCT116, HeLa-S3, LNCAP, Panc1, RWPE1, 22Rv1, VCaP
characterized to standards
Homo sapiens
GM23338, bipolar neuron
characterized to standards with exemption
Mus musculus
liver, lung, midbrain, hindbrain, heart, kidney, stomach, forebrain, intestine, limb
characterized to standards with exemption
- Status
- released
- Source (vendor)
- Active Motif
- Product ID
- 61311
- Lot ID
- 23913002
- Characterized targets
- CTCF (Homo sapiens), CTCF (Mus musculus)
- Host
- rabbit
- Clonality
- polyclonal
- Purification
- affinity
- Isotype
- IgG
- Antigen description
- Peptide within the N-terminal region of human CTCF
- External resources
Characterizations
CTCF (Homo sapiens)
22Rv1RWPE1VCaP
compliant
- Caption
- Western blot analysis of nuclear lysates prepared from multiple cells lines loaded in the order: 22Rv1,RWPE-1,VCaP using the antibody 61311. Molecular Weight: 120.
- Submitted by
- Nathaniel Watson
- Lab
- Michael Snyder, Stanford
- Grant
- U54HG006996
- Download
- CTCF_AM_WB_Results_2.png
CTCF (Homo sapiens)
C4-2BGM12878HCT116HeLa-S3HepG2LNCAPK562MCF-7Panc1RWPE1
compliant
- Caption
- Western blot analysis of nuclear lysates prepared from multiple cells lines loaded in the order: C4-2B,GM12878,HCT116,HeLa-S3,HepG2,LNCaP,K562,MCF-7,Panc1,RWPE-1 using the antibody 61311. Molecular Weight: 120.
- Submitted by
- Nathaniel Watson
- Lab
- Michael Snyder, Stanford
- Grant
- U54HG006996
- Download
- CTCF_AM_WB_Results_WB-1016.png
CTCF (Homo sapiens)
K562
not submitted for review by lab
- Reviewer comment
- IP gel for mass spectrometry analysis
- Submitted by
- Mark Mackiewicz
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- CTCF_IP-Commassie.png
CTCF (Homo sapiens)
K562GM12878HepG2MCF-7
exempt from standards
- Caption
- Whole cell lysates of K562, GM12878, HepG2, and MCF7 were immunoprecipitated using the primary antibody (Active Motif; 61311). The IP fraction was separated on a 12% acrylamide gel with the Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. The membrane was probed with the primary antibody (same as that used for IP) and a secondary HRP-conjugated antibody. The resulting bands were visualized with SuperSignal West Femto Solution (Thermo Scientific). Protein Marker (PM) is labeled in kDa. The approximate size of CTCF is ~83 kDa.
- Submitter comment
- --
- Reviewer comment
- Band of interest is not 50% of overall signal in lane. Rescued by mass spectrometry analysis
- Submitted by
- Mark Mackiewicz
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- CTCF_IP-WB.png
CTCF (Homo sapiens)
compliant
- Caption
- K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61311). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. Gel fragments (rectangle outline) corresponding to the bands indicated on the Coomassie Blue stained gel image were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of gel fragment 1 from K562: The sample was analyzed on a LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectral analysis with probability based matching at p < 0.05. SEQUEST results were reported with ProteinProphet protXML Viewer (TPP v4.4 JETSTREAM) and filtered for a minimum probability of 0.9. All protein hits that met these criteria were reported, including common contaminants. Fold enrichment for each protein reported was determined using a custom script based on the FC-B score calculation from the reference Mellacheruvu et al., 2013. The CRAPome: a contaminant repository for affinity purification mass spectrometry data. Nat. Methods. 10(8):730-736. Doi:10.1038/nmeth.2557. The target protein, CTCF, was identified as the 1st enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
- Submitted by
- Mark Mackiewicz
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- CTCF_K562-1.png
CTCF (Homo sapiens)
not compliant
- Caption
- K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61311). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. Gel fragments (rectangle outline) corresponding to the bands indicated on the Coomassie Blue stained gel image were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of gel fragment 2 from K562: The sample was analyzed on a LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectral analysis with probability based matching at p < 0.05. SEQUEST results were reported with ProteinProphet protXML Viewer (TPP v4.4 JETSTREAM) and filtered for a minimum probability of 0.9. All protein hits that met these criteria were reported, including common contaminants. Fold enrichment for each protein reported was determined using a custom script based on the FC-B score calculation from the reference Mellacheruvu et al., 2013. The CRAPome: a contaminant repository for affinity purification mass spectrometry data. Nat. Methods. 10(8):730-736. Doi:10.1038/nmeth.2557. The target protein, CTCF, was not identified by IP-Mass Spectrometry.
- Submitted by
- Mark Mackiewicz
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- CTCF_K562-2.png
CTCF (Homo sapiens)
not compliant
- Caption
- K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61311). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. Gel fragments (rectangle outline) corresponding to the bands indicated on the Coomassie Blue stained gel image were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of gel fragment 3 from K562: The sample was analyzed on a LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectral analysis with probability based matching at p < 0.05. SEQUEST results were reported with ProteinProphet protXML Viewer (TPP v4.4 JETSTREAM) and filtered for a minimum probability of 0.9. All protein hits that met these criteria were reported, including common contaminants. Fold enrichment for each protein reported was determined using a custom script based on the FC-B score calculation from the reference Mellacheruvu et al., 2013. The CRAPome: a contaminant repository for affinity purification mass spectrometry data. Nat. Methods. 10(8):730-736. Doi:10.1038/nmeth.2557. The target protein, CTCF, was not identified by IP-Mass Spectrometry.
- Submitted by
- Mark Mackiewicz
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- CTCF_K562-3.png
CTCF (Homo sapiens)
compliant
- Caption
- The motif for target CTCF is represented by the attached position weight matrix (PWM) derived from file ENCFF030LLP. Motif enrichment analysis was done by Dr. Zhizhuo Zhang (Broad Institute, Kellis Lab) using known motifs (http://compbio.mit.edu/encode-motifs/) and previously published ChIP-seq data (http://www.broadinstitute.org/~zzhang/motifpipeline/data/TrainSetInfo.txt). The accept probability score of the given transcription factor was calculated using a Bayesian approach. This analysis also includes three motif enrichment scores, computed by overlapping the motif instances with the given ChIP-seq peak locations. For more information on the underlying statistical methods, please see the attached document. Accept probability score: 0.987646461 Global enrichment Z-score: 12.820532684 Positional bias Z-score: 14.0533855528 Peak rank bias Z-score: 5.50975231904, Enrichment rank: 1.0.
- Submitted by
- Aditi Narayanan
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- ENCAB247TYO.pdf
CTCF (Homo sapiens)
GM23338bipolar neuron
exempt from standards
- Caption
- The ENCODE Binding Working Group finds for some valuable biosamples that recreating a primary on well-characterized antibodies is not cost effective. Therefore, they allow exemption from standards for these samples.
- Submitter comment
- The lab is asking for an exemption forIPSCs due to the lack of resources to make a primary characterization for them
- Reviewer comment
- Exempted by the Feb 29, 2016 antibody review panel
- Submitted by
- Richard Myers
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- No_biosample.png
CTCF (Mus musculus)
liverlungmidbrainhindbrainheartkidneystomachforebrainintestinelimb
exempt from standards
- Caption
- The ENCODE Binding Working Group finds for some valuable tissues that recreating a primary on well characterized antibodies is not cost effective. Therefore, they allow exemption from standards for these tissues.
- Submitter comment
- The lab is asking for an exemption for several tissue cells due to the lack of resource to make a primary characterization for them
- Reviewer comment
- Exempted by the Feb 29, 2016 antibody review panel
- Submitted by
- Mark Mackiewicz
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- No_tissue.png