ENCAB247TYO

Antibody against Mus musculus CTCF, Homo sapiens CTCF

Homo sapiens
MCF-7, Panc1, K562, HeLa-S3, GM12878, RWPE1, LNCAP, HCT116, HepG2, C4-2B, VCaP, 22Rv1
characterized to standards
Mus musculus
forebrain, kidney, stomach, heart, hindbrain, limb, intestine, midbrain, lung, liver
characterized to standards with exemption
Homo sapiens
GM23338, bipolar neuron
Status
released
Source (vendor)
Active Motif
Product ID
61311
Lot ID
23913002
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
Method: immunoblot
Attachment from submitter
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
CTCF (Homo sapiens)
C4-2BGM12878HCT116HeLa-S3HepG2LNCAPK562MCF-7Panc1RWPE1
Method: immunoblot
Attachment from submitter
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
CTCF (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Reviewer comment
IP gel for mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
CTCF (Homo sapiens)
K562GM12878HepG2MCF-7
Method: immunoblot
Attachment from submitter
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
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CTCF (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
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
Download
CTCF (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
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
Download
CTCF (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
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
Download
CTCF (Homo sapiens)
Method: motif enrichment
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
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CTCF (Homo sapiens)
GM23338bipolar neuron
Method: immunoblot
Attachment from submitter
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
CTCF (Mus musculus)
liverlungmidbrainhindbrainheartkidneystomachforebrainintestinelimb
Method: immunoblot
Attachment from submitter
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
Download