ENCAB000AUF

Antibody against Homo sapiens TARDBP

Homo sapiens
GM12878, K562
characterized to standards
Homo sapiens
HepG2
characterized to standards with exemption
Homo sapiens
any cell type or tissue
partially characterized
Status
released
Source (vendor)
GeneTex
Product ID
GTX114210
Lot ID
40135
Characterized targets
TARDBP (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Antigen description
Recombinant fgmt within amino acids 1 and 289 of TARDBP (Uniprot ID#Q13148)
External resources

Characterizations

TARDBP (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
not 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, TARDBP, was not identified as an enriched protein in band 1 based on IP-Mass Spectrometry.
Reviewer comment
TARDBP does not show up in the list at all
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
TARDBP (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
exempt from standards
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, TARDBP, was identified as the 27th enriched protein and the 2nd ranked transcription factor in band 2 based on IP-Mass Spectrometry.
Submitter comment
Although this is the 27th ranked peptide it is the 2nd ranked transcription factor
Reviewer comment
As per the decisions of the Antibody Review Panel on Feb 29, 2016. This characterization is exempted from standards.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
TARDBP (Homo sapiens)
GM12878
Method: immunoprecipitation
Attachment from submitter
Reviewer comment
IP gel for mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TARDBP (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
GM12878 whole cell lysate was immunoprecipitated using the primary antibody (GeneTex; GTX114210). 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 GM12878: 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, TARDBP, was identified as the 1st enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TARDBP (Homo sapiens)
K562GM12878
Method: immunoblot
Attachment from submitter
compliant
Caption
Whole cell lysates of K562 and GM12878 were immunoprecipitated using the primary antibody (GeneTex; GTX114210). 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 ~42 kDa.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TARDBP (Homo sapiens)
Method: immunoprecipitation
Attachment from submitter
not reviewed
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (GeneTex; GTX114210). 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.
Submitter comment
This image is to show which bands are used for the mass spec
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TARDBP (Homo sapiens)
K562HepG2
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of K562 and HepG2 were immunoprecipitated using the primary antibody (GeneTex; GTX114210). 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 ~35 and ~45 kDa.
Submitter comment
These characterizations were done during ENCODE2 when there was no requirement for IgG control.
Reviewer comment
As per Antibody review panal decsion of Feb 29, 2016, this will be exempted from standards
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB