- Term name
- Term ID
- Homo sapiens HepG2 cell line genetically modified (insertion) using CRISPR targeting H. sapiens SMAD3
immunoprecipitation followed by mass spectrometry
- SMAD3 IP-MS. 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, SMAD3, was identified as the 1st enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
- Submitted by
- Scott Newberry
- Richard Myers, HAIB