1. Antibodies
  2. Homo sapiens
  3. TFAP4

ENCAB000BJE

Antibody against Homo sapiens TFAP4

Homo sapiens
K562, HepG2
characterized to standards
Status
released
Source (vendor)
Sigma
Product ID
WH0007023M3
Lot ID
07040-7A10
Characterized targets
TFAP4 (Homo sapiens)
Host
mouse
Clonality
monoclonal
Isotype
IgG2aκ
Antigen description
TFAP4 (NP_003214, a.a. 93-193) partial recomb protein with GST tag. MW of GST tag alone is 26 kDa.
External resources

Characterizations

TFAP4 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
compliant
Caption
HepG2 whole cell lysate was immunoprecipitated using the primary antibody (Sigma; WH0007023M3). 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 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, TFAP4, was identified as the 2nd ranked enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Reviewer comment
Within top 20 enriched protein ranked by fold enrichment
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
 TFAP4-H-1.pdf
TFAP4 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
not compliant
Caption
HepG2 whole cell lysate was immunoprecipitated using the primary antibody (Sigma; WH0007023M3). 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 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, TFAP4, was identified as the 64th ranked enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Reviewer comment
Not within top 20 factors ranked by fold enrichment
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
 TFAP4-H-2.pdf
TFAP4 (Homo sapiens)
HepG2
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
not reviewed
Caption
Whole cell lysates of HepG2 were immunoprecipitated using the primary antibody (Sigma; WH0007023M3). 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 ~46 and 49 kDa.
Reviewer comment
IP gel for mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
TFAP4 (Homo sapiens)
K562HepG2
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
compliant
Caption
An immunoprecipitation followed by Western blot was performed with Sigma WH0007023M3 (lot 07040-7A10) antibody against TFAP4 in K562 and HepG2 whole cell lysate along with a control IP with normal IgG from the same host species as the test antibody (mouse, Santa Cruz sc-2025). The expected size of TFAP4 is 38 kDa. A band of ~40-48 kDa was detected in the test IP and not in the control IP. The experiment was repeated and the band was analyzed by mass spec in the secondary validation.
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB