ENCAB000BQG

Antibody against Homo sapiens JUNB

Homo sapiens
K562, induced pluripotent stem cell, GM12878, GM23338
characterized to standards with exemption
Homo sapiens
any cell type or tissue
partially characterized
Status
released
Source (vendor)
Active Motif
Product ID
39550
Lot ID
34708001
Characterized targets
JUNB (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
crude
Isotype
serum
Antigen description
antibody was raised against a synthetic peptide corresponding to amino acids 28-42 of human JunB
External resources

Characterizations

JUNB (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
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, JUNB, was identified as the 9th ranked enriched protein and the 1st ranked transcription factor in gel fragment 1 based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
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JUNB (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
not compliant
Caption
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, JUNB, was identified as the 72nd ranked enriched protein and the 3rd ranked transcription factor in gel fragment 2 based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
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JUNB (Homo sapiens)
Method: immunoprecipitation
Attachment from submitter
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 39550). 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 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.
Submitter comment
Gel for mass spectrometry characterization
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
JUNB (Homo sapiens)
K562GM12878GM23338
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of K562 and GM12878 were immunoprecipitated using the primary antibody (Active Motif; 39550). 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 ~42 and ~46 kDa.
Submitter comment
The lab is asking for an exemption for IPS cells due to the lack of resource to make a primary characterization for them
Reviewer comment
Major band is not within 20% of expected size. Rescued by mass spectrometry. IPS cells accepted via exemption from the Binding Group.
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB
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JUNB (Homo sapiens)
induced pluripotent stem cell
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