ENCAB000AID

Antibody against Mus musculus JUND, Homo sapiens JUND

Homo sapiens
liver, HepG2
characterized to standards with exemption
Mus musculus
any cell type or tissue
partially characterized
Homo sapiens
K562
not characterized to standards
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-74
Lot ID
unknown
Host
rabbit
Clonality
polyclonal
Isotype
IgG

Characterizations

JUND (Homo sapiens)
K562
Method: immunoblot
not compliant
Reviewer comment
WB image not in .pdf -MHO
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
JUND (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
not compliant
Caption
IP followed by mass spectrometry: Briefly, K562 whole cell lysates were immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. Gel was stained with Coomassie Blue in order to visualize marker bands. Gel fragments corresponding to the bands indicated above in the western blot image were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. There the samples were run on an LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectra analysis, with probability based matching at p < 0.05. As per ENCODE data standards, all SEQUEST results are attached (ENCODE_HAIB_YYY_sc77742_09262011_MassSpec.pdf), including common contaminants. Target protein is listed as hit 2a in the ~90kDa band.
Reviewer comment
Cannot review without gel.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
JUND (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
HepG2 whole cell lysate was immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-74). 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 HepG2: 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, JUND, was identified as the 17th ranked enriched protein and the 2nd ranked transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
JUND (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
HepG2 whole cell lysate was immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-74). 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 HepG2: 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, JUND, was identified as the 14th ranked enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
JUND (Mus musculus)
Method: immunoprecipitation
Attachment from submitter
Submitter comment
Gel for mass spec analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
JUND (Homo sapiens)
HepG2
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of HepG2 were immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-74). 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. Two bands were detected at ~39 and 45 kDa.
Submitter comment
--
Reviewer comment
Band of interest is not 50% of overall signal in lane (barely), but rescued by mass spec
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
JUND (Homo sapiens)
Method: motif enrichment
Attachment from submitter
compliant
Caption
The motif for target JUND is represented by the attached position weight matrix (PWM) derived from files ENCFF734AWV and ENCFF444QCD. 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. From ENCFF444QCDL: Accept probability score: 0.82839478, Global enrichment Z-score: 6.69834273714, Positional bias Z-score: 11.8147584565, Peak rank bias Z-score: 11.8419168095. From ENCFF734AVW: Accept probability score: 0.82839478, Global enrichment Z-score: 6.77610046879, Positional bias Z-score: 12.3000727697, Peak rank bias Z-score: 11.2991712854, Enrichment rank: 1.0.
Submitted by
Aditi Narayanan
Lab
Richard Myers, HAIB
JUND (Homo sapiens)
liver
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 liver 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
Richard Myers
Lab
Richard Myers, HAIB
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