ENCAB000ALM

Antibody against Homo sapiens TAF1

Homo sapiens
GM12878, K562, liver, HeLa-S3
characterized to standards with exemption
Homo sapiens
HepG2
partially characterized
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-735
Lot ID
K0905
Characterized targets
TAF1 (Homo sapiens)
Lot ID aliases
G2909, F2706
Host
mouse
Clonality
monoclonal

Characterizations

TAF1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
not compliant
Caption
IP followed by mass spectrometry: Briefly, K562 whole cell lysates were immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. Gel was stained with Coomassie Blue in order to visualize marker bands. A gel fragment corresponding to the band indicated above in the western blot image was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. There the sample was run on an LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectra analysis, with probability based matching at p < 0.05. As per ENCODE data standards, all SEQUEST results are attached (ENCODE_HAIB_TAF1_03172011_MassSpec.pdf), including common contaminants. Target protein is listed as hit 5a.
Reviewer comment
Missing mass spec data
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
TAF1 (Homo sapiens)
liver
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
The ENCODE Binding Working Group finds for some valuable tissues that recreating a primary on well characterized antibodies is not cost effective. Therefore, they allow exemption from standards for these tissues.
Submitter comment
The lab is asking for an exemption for liver cells due to the lack of resource to make a primary characterization for them
Reviewer comment
Exempted by the Feb 29, 2016 antibody review panel
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
Download
TAF1 (Homo sapiens)
K562GM12878HepG2HeLa-S3
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
Western blot protocol: Whole cell lysate was immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. Blotting with primary (same as that used for IP) and secondary HRP-conjugated antibodies was performed on an Invitrogen BenchPro 4100 system. Visualization was achieved using SuperSignal West Femto solution (Thermo Scientific). Results: Band of expected size visualized, representing strongest signal in the lane. Figure legend: IP-western with sc-735 in whole cell lysates (WCL) of K562, GM12878, HepG2, and HeLa; PM=protein marker. TAF1 band is indicated.
Submitter comment
We'd like an exemption for this characterization.
Reviewer comment
The antibody review panel has decided that antibodies that pass ENCODE2 standards, and are only missing their IgG controls for ENCODE3 standards, will be passed via exemption. In this case, HepG2 does not pass ENCODE2 standards as the band is not 50% of the overall signal
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
TAF1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
not compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-735). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. Two bands at ~216 and ~250 kDa were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of Band 1 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, TAF1, was identified as the 47th enriched protein and the 4th transcription factor based on IP-Mass Spectrometry.
Reviewer comment
Factor of interest is not within the top 20 proteins ranked by fold enrichment
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
TAF1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-735). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. Two bands at ~216 and ~250 kDa were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of Band 2 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, TAF1, was identified as the 16th enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
TAF1 (Homo sapiens)
K562GM12878HepG2HeLa-S3
Method: immunoprecipitation
Attachment from submitter
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download