ENCAB000AAG

Antibody against Homo sapiens SMAD5

Homo sapiens
GM12878, K562
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
Method: immunoblot
Attachment from submitter
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
SMAD5 (Homo sapiens)
K562HepG2
Method: immunoprecipitation
Attachment from submitter
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
SMAD5 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
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