ENCAB000ACR

Antibody against Homo sapiens TCF7

Homo sapiens
HepG2, GM12878, K562
characterized to standards with exemption
Homo sapiens
any cell type or tissue
partially characterized
Status
released
Source (vendor)
Sigma
Product ID
WH0006932M1
Lot ID
11181-1D2
Characterized targets
TCF7 (Homo sapiens)
Host
mouse
Clonality
monoclonal
Isotype
IgG1κ
Antigen description
TCF7 (NP_003193, a.a. 287-385) partial recombinant protein with GST tag.
External resources

Characterizations

TCF7 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
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, TCF7, was identified as the 1st ranked enriched protein and the 1st ranked transcription factor in band 1 based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TCF7 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
Analysis of gel fragment 2 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, TCF7, was identified as the 10th ranked enriched protein and the 1st ranked transcription factor in band 2 based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TCF7 (Homo sapiens)
K562GM12878HepG2
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of K562, MCF7, and GM12878 were immunoprecipitated using the primary antibody (Active Motif; 39548). 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 ~47 kDa.
Submitter comment
--
Reviewer comment
Double band, but any issues rescued by mass spec.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TCF7 (Homo sapiens)
Method: immunoprecipitation
Attachment from submitter
not reviewed
Caption
GM12878 whole cell lysate was immunoprecipitated using the primary antibody (Sigma; WH0006932M1). 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.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TCF7 (Homo sapiens)
K562GM12878
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of K562 and GM12878 were immunoprecipitated using the primary antibody (Sigma; WH0006932M1). 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 50 kDa.
Submitter comment
the standards allow for using mass spec to rescue an immunoblot
Reviewer comment
There bands to not reflect the size of the TF, but the mass spec shows that it is there.
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB
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