1. Antibodies
  2. Homo sapiens
  3. KDM4B

ENCAB000BQH

Antibody against Homo sapiens KDM4B

Homo sapiens
K562, HepG2
characterized to standards with exemption
Status
released
Source (vendor)
Active Motif
Product ID
61222
Lot ID
34711001
Characterized targets
KDM4B (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
affinity
Isotype
IgG
Antigen description
antibody was raised against a peptide within the C-terminal region of human JMJD2B.
External resources

Characterizations

KDM4B (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
Attachment from submitters
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61222). 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, KDM4B, 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
 KDM4B-1.png
KDM4B (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
Attachment from submitters
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61222). 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, KDM4B, was identified as the 2nd enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
 KDM4B-2.png
KDM4B (Homo sapiens)
K562HepG2
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
exempt from standards
Caption
Whole cell lysates of K562 and HepG2 were immunoprecipitated using the primary antibody (Active Motif; 61222). 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 ~57 and 160 kDa.
Submitter comment
--
Reviewer comment
Non specific bands, but rescued by mass spec analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
 KDM4B_IP-WB.png
KDM4B (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
not reviewed
Reviewer comment
IP gel for mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB