ENCAB697XQW

Antibody against synthetic tag FLAG, synthetic tag 3xFLAG

Homo sapiens
K562, HepG2, MCF-7, SK-N-SH
characterized to standards
Status
released
Source (vendor)
Sigma
Product ID
F1804
Lot ID
SLBK1346V
Host
mouse
Clonality
monoclonal
Purification
affinity
Isotype
IgG1
Antigen description
Recognizes the FLAG peptide sequence: DYKDDDDK
External resources

Characterizations

3xFLAG-ZNF124 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
HepG2 whole cell lysate was immunoprecipitated using the primary antibody (Sigma; F1804). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. The whole lane was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of whole lane gel 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, ZNF124, 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
FLAG (synthetic tag)
K562HepG2MCF-7
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
We take the whole lane for IP-MS as opposed to cutting out specific bands. This is an image of what we took as a whole lane for some standard antibody immunoprecipitations. The black boxes represent the fact that we cut the whole lane, we do not run out the gel to get specific bands.
Submitter comment
We believe that the mass spec is enough to characterize this antibody and would apply the mass spec rescue scenario described in the standards document.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB