ENCAB000ATW

Antibody against Homo sapiens ZHX2

Homo sapiens
K562
characterized to standards
Homo sapiens
HepG2, GM12878, MCF-7
characterized to standards with exemption
Status
released
Source (vendor)
GeneTex
Product ID
GTX112232
Lot ID
40107
Characterized targets
ZHX2 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Isotype
IgG
Antigen description
Recombinant fgmt within amino acids 533 and 826 of ZHX2 (Uniprot ID#Q9Y6X8)
External resources

Characterizations

ZHX2 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
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, ZHX2, was identified as the 1st ranked enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Reviewer comment
Rescues failed immunoblot.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
ZHX2 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
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, ZHX2, was identified as the 1st ranked enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Reviewer comment
Rescues failed immunoblot.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
ZHX2 (Homo sapiens)
HepG2
Method: immunoprecipitation
Attachment from submitter
not reviewed
Caption
HepG2 whole cell lysate was immunoprecipitated using the primary antibody (GeneTex; GTX112232). 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.
Submitter comment
None
Reviewer comment
Missing IgG Control. Submitted as gel for the secondary mass spec characterization.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
ZHX2 (Homo sapiens)
GM12878HepG2MCF-7
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of GM12878, HepG2, and MCF7 were immunoprecipitated using the primary antibody (GeneTex; GTX112232). 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 ~53 and ~117kDa.
Submitter comment
--
Reviewer comment
Band of interest is not 50% of overall signal in lane, is a doublet, and in some cases, appears slightly in control lane. Rescued by mass spec.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
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ZHX2 (Homo sapiens)
K562HepG2
Method: immunoblot
Attachment from submitter
compliant
Caption
Whole cell lysates of K562 and HepG2 were immunoprecipitated using the primary antibody (GeneTex; GTX112232). 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 ~43 and ~100kDa.
Submitter comment
None
Reviewer comment
Hep G2 band not 50% of signal, but rescued by mass spec. GM12878 is compliant
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB
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