1. Antibodies
  2. Homo sapiens
  3. ZBTB33

ENCAB000AML

Antibody against Homo sapiens ZBTB33

Homo sapiens
K562, GM12878, HeLa-S3, HepG2, liver
characterized to standards with exemption
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-23871
Lot ID
10308
Characterized targets
ZBTB33 (Homo sapiens)
Host
mouse
Clonality
monoclonal
Isotype
IgG1
Antigen description
Kaiso (6F8) was Raised against amino acids 1-504 representing full length Kaiso of human origin.

Characterizations

ZBTB33 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
not compliant
Caption
Briefly, K562 whole cell lysates were immunoprecipitated using sc-98589 (the antibody used to detect ZBTB33 band on the IP-western), 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 MASCOT tandem mass spectra analysis, with probability based matching at p < 0.05. As per ENCODE data standards, all MASCOT results are attached, including common contaminants. Target protein is listed as hit 20 (row 21 in file).
Reviewer comment
Not compliany by ENCODE2 or 3 standards, and data is not presented clearly.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
ZBTB33 (Homo sapiens)
K562GM12878HeLa-S3HepG2
Method: immunoblot
Attachment from submitter
Attachment from submitters
exempt from standards
Caption
Western blot protocol: Whole cell lysate was immunoprecipitated using primary antibody (sc-23871), 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 was performed with sc-98589 Kaiso (H-154) antibody, followed by secondary HRP-conjugated antibody, 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-23871 (IP) and sc-98589 (detection) in whole cell lysates (WCL) of K562, GM12878, HepG2, and HeLa; PM=protein marker. ZBTB33 band is indicated.
Submitter comment
The lab is asking for an exemption for this characterization because it was done before the agreements to have an IgG control
Reviewer comment
As per Antibody review panal decsion of Feb 29, 2016, this will be exempted from standards
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
ZBTB33 (Homo sapiens)
liver
Method: immunoblot
Attachment from submitter
Attachment from submitters
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
 No_tissue.png
ZBTB33 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
not submitted for review by lab
Reviewer comment
IP gel for mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
ZBTB33 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
Attachment from submitters
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-23871). 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. 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, ZBTB33, was identified as the 10th enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB