ENCAB000AAG
Antibody against Homo sapiens SMAD5
Homo sapiens
K562, GM12878
characterized to standards with exemption
Homo sapiens
HepG2
partially characterized
- Status
- released
- Source (vendor)
- Sigma
- Product ID
- WH0004090M1
- Lot ID
- 10267-2D7
- Characterized targets
- SMAD5 (Homo sapiens)
- Host
- mouse
- Clonality
- monoclonal
- Isotype
- IgG2aκ
- Antigen description
- SMAD5 (AAH09682, a.a. 105-182) partial recombinant protein with GST tag.
- External resources
Characterizations
SMAD5 (Homo sapiens)
K562GM12878
exempt from standards
- Caption
- Whole cell lysates of K562 and HepG2 were immunoprecipitated using the primary antibody (Sigma; WH0004090M1). 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 ~55 kDa.
- Submitter comment
- Band is not 50% of signal, rescued by mass spec.
- Reviewer comment
- Band is not 50% of overall signal, but rescued by mass spec.
- Submitted by
- Flo Pauli-Behn
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- SMAD5_Immunoblot.png
SMAD5 (Homo sapiens)
K562HepG2
not reviewed
- Caption
- K562 whole cell lysate was immunoprecipitated using the primary antibody (Sigma; WH0004090M1). 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
- Band not 50% of signal and lacks IgG control, but is mostly supplementary to mass spec rescue of the immunoblot
- Reviewer comment
- Band not 50% of signal, but rescued by mass spec
- Submitted by
- Flo Pauli-Behn
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- SMAD5_IP_MS_WB.png
SMAD5 (Homo sapiens)
compliant
- Caption
- Analysis of gel fragment 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, SMAD5, was identified as the 2nd ranked enriched protein and the 2nd ranked transcription factor based on IP-Mass Spectrometry.
- Submitter comment
- Rescues immunoblot and IP
- Submitted by
- Collin White
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- SMAD5_Mass spec.png