1. Antibodies
  2. Homo sapiens
  3. HNF4A

ENCAB000AHP

Antibody against Homo sapiens HNF4A

Homo sapiens
HepG2, liver
characterized to standards with exemption
Homo sapiens
K562
not characterized to standards
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-8987
Lot ID
G1309
Characterized targets
HNF4A (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Isotype
IgG
Antigen description
Epitope corresponding to a.a. 295-465 mapping at the C-terminus of HNF4A of human origin.

Characterizations

HNF4A (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
not compliant
Caption
ENCODE data standards recognizes various methodologies for secondary validation of antibodies. Among these methodologies is immunoprecipitation followed by mass spectrometry analysis. Briefly, HepG2 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 Coomasie Blue in order to visualize marker bands. A gel fragment corresponding to the band indicated above in the western blot image at ~55 kDa and a second fragment at ~38 kDa were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. There the samples were run on an LTQ XL Linear Ion Trap Mass Spectrometer with alternating collision-induced dissociation and electron-transfer dissociation. Peptides were identified using MASCOT (Matrix Science), with probability based matching at p < 0.05. Subsequent analysis was performed in Scaffold (Proteome Software, Inc.) at 0.0% protein FDR and 1.7% peptide FDR. As per ENCODE data standards, all Scaffold results are listed below, including common contaminants. Target protein is highlighted in bold font.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
HNF4A (Homo sapiens)
HepG2K562
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
exempt from standards
Caption
Whole cell lysates or nuclear extracts 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. 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).
Submitter comment
We'd like an exemption for this characterization.
Reviewer comment
The antibody review panel will pass (with exemption) any immunoprecipitation characterization that meets all standards for ENCODE2. ENCODE2 did not require an IgG control
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
HNF4A (Homo sapiens)
Method: motif enrichment
Attachment from submitter
Attachment from submitters
compliant
Caption
The motif for target HNF4A is represented by the attached position weight matrix (PWM) derived from ENCFF922YKH and ENCFF703MBR. Motif enrichment analysis was done by Dr. Zhizhuo Zhang (Broad Institute, Kellis Lab) using known motifs (http://compbio.mit.edu/encode-motifs/) and previously published ChIP-seq data (http://www.broadinstitute.org/~zzhang/motifpipeline/data/TrainSetInfo.txt). The accept probability score of the given transcription factor was calculated using a Bayesian approach. This analysis also includes three motif enrichment scores, computed by overlapping the motif instances with the given ChIP-seq peak locations. For more information on the underlying statistical methods, please see the attached document. From ENCFF922YKH: Accept probability score: 0.980692270556, Global enrichment Z-score: 7.73224741061, Positional bias Z-score: 9.73903092699, Peak rank bias Z-score: 7.9240626667. From ENCFF703MBR: Accept probability score: 0.980692270556, Global enrichment Z-score: 8.40991465285, Positional bias Z-score: 10.0966485197, Peak rank bias Z-score: 6.9130845021, Enrichment rank: 4.0.
Submitted by
Aditi Narayanan
Lab
Richard Myers, HAIB
HNF4A (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