ENCAB000BLM

Antibody against Homo sapiens NRF1

Homo sapiens
GM12878, K562, HepG2, HeLa-S3, MCF-7, liver
characterized to standards
Status
released
Source (vendor)
CDI
Product ID
R157.1.3H3
Lot ID
011613.RAP
Characterized targets
NRF1 (Homo sapiens)
Host
mouse
Clonality
monoclonal
Purification
affinity
Isotype
IgG2a
External resources

Characterizations

NRF1 (Homo sapiens)
Method: motif enrichment
compliant
Caption
The motif for target NRF1 is represented by the attached position weight matrix (PWM) derived from file ENCFF664FFU. 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: 0.919608216391, Global enrichment Z-score: 6.54210849562, Positional bias Z-score: 13.5920351772, Peak rank bias Z-score: 14.9637561882, Enrichment rank: 1.0.
Submitted by
Aditi Narayanan
Lab
Richard Myers, HAIB
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NRF1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (CDI; R157.1.3H3). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. A gel fragment (rectangle outline) corresponding to the band indicated on the Coomassie Blue stained gel image was 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, NRF1, was identified as the 6th ranked enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
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NRF1 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Caption
Whole cell lysates of K562 were immunoprecipitated using the primary antibody (CDI; R157.1.3H3). 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. One band was detected at ~73 kDa. Expected size is ~53.5 kDa.
Submitter comment
--
Reviewer comment
IP gel for mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
NRF1 (Homo sapiens)
GM12878K562HepG2HeLa-S3MCF-7liver
Method: immunoblot
Attachment from submitter
compliant
Caption
a) Western blot analysis of nuclear lysates prepared from multiple cells lines loaded in the order: GM12878, K562, HepG2, HelaS3, MCF7, Liver using the antibody R157.1.3H3. Molecular mass: 53541 Da
Reviewer comment
Multiple bands, blob near expected size of 53kD/46kD (not indicated on blot or caption).
Submitted by
Trupti Kawli
Lab
Michael Snyder, Stanford
NRF1 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
not compliant
Caption
b) Immunoprecipitation was performed on nuclear extracts from the cell line: K562, using the antibody R157.1.3H3 .The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG. Expected size ~55 kDa.
Reviewer comment
Band not within 20% of expected size
Submitted by
Trupti Kawli
Lab
Michael Snyder, Stanford