ENCAB000AJM

Antibody against Mus musculus EP300, Homo sapiens EP300

Mus musculus
limb, intestine, heart, stomach, midbrain, lung, liver, forebrain, kidney, hindbrain
characterized to standards with exemption
Homo sapiens
neural cell, HeLa-S3, GM12878, K562, HepG2
Homo sapiens
any cell type or tissue
partially characterized
Mus musculus
any cell type or tissue
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-585
Lot ID
D0411
Host
rabbit
Clonality
polyclonal

Characterizations

EP300 (Homo sapiens)
Method: immunoblot
Attachment from submitter
not reviewed
Caption
Western blot protocol: Whole cell lysates were immunoprecipitated using primary antibody (sc-585), 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-585 in WCL (whole cell lysates) of K562, GM12878, HepG2, and HeLa; PM=protein marker. p300 band is indicated.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
EP300 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
GM12878 whole cell lysate was immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-585). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. The whole lane was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of whole lane from GM12878: 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, EP300, was identified as the 1st enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitter comment
In some cases, we exercised the option of extracting the whole lane to send for IP-mass spectrometry as opposed to cutting and sending discrete bands from a gel. This is especially the case when the antibody results in multiple and non-discrete bands or no visible bands in the predicted size range of the protein when observed by IP-Western. To ensure that the targeted protein can be identified in the IP, whole cell lysates are loaded in a 12% Bio-Rad TGX gel and run for 5-10 minutes using the Bio-Rad Tetra Cell system. The entire gel lane or just the single unseparated band is excised from the gel and sent for analysis. We do not provide coomassie blue-stained gel images in these instances as all one observes is a thick, unresolved band near the loading well.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
EP300 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
HepG2 whole cell lysate was immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-585). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. The whole lane was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of whole lane from HepG2: 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, EP300, was identified as the 2nd enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitter comment
In some cases, we exercised the option of extracting the whole lane to send for IP-mass spectrometry as opposed to cutting and sending discrete bands from a gel. This is especially the case when the antibody results in multiple and non-discrete bands or no visible bands in the predicted size range of the protein when observed by IP-Western. To ensure that the targeted protein can be identified in the IP, whole cell lysates are loaded in a 12% Bio-Rad TGX gel and run for 5-10 minutes using the Bio-Rad Tetra Cell system. The entire gel lane or just the single unseparated band is excised from the gel and sent for analysis. We do not provide coomassie blue-stained gel images in these instances as all one observes is a thick, unresolved band near the loading well.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
EP300 (Homo sapiens)
K562GM12878HepG2
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of K562, GM12878, and HepG2 were immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-585). 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. The approximate size of EP300 is ~264 kDa, although no distinguishable band was observed at this size range.
Submitter comment
--
Reviewer comment
Band of interest is not 50% of overall signal in lane. Rescued by mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
EP300 (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-585). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. The whole lane was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of whole lane 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, EP300, was identified as the 1st enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitter comment
In some cases, we exercised the option of extracting the whole lane to send for IP-mass spectrometry as opposed to cutting and sending discrete bands from a gel. This is especially the case when the antibody results in multiple and non-discrete bands or no visible bands in the predicted size range of the protein when observed by IP-Western. To ensure that the targeted protein can be identified in the IP, whole cell lysates are loaded in a 12% Bio-Rad TGX gel and run for 5-10 minutes using the Bio-Rad Tetra Cell system. The entire gel lane or just the single unseparated band is excised from the gel and sent for analysis. We do not provide coomassie blue-stained gel images in these instances as all one observes is a thick, unresolved band near the loading well.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
EP300 (Mus musculus)
HeLa-S3
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
The ENCODE antibody standards document exempts some valuable and/or limiting samples from primary characterizations for well-characterized antibodies. They are given exemptions so that the samples can be conserved to carry out the downstream experiments.
Submitter comment
Submitting for Peggy Farnham to affect Snyder experiments.
Reviewer comment
Submitting for Peggy Farnham to affect Snyder experiments.
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
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EP300 (Homo sapiens)
neural cell
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
The ENCODE antibody standards document exempts some valuable and/or limiting samples from primary characterizations for well-characterized antibodies. They are given exemptions so that the samples can be conserved to carry out the downstream experiments.
Submitter comment
We do not have any of the H1-derived neurons that were distributed to the consortium for analysis in ENCODE2 left.
Reviewer comment
This cell type was exempted from primary characterization of antibody ENCAB000AIT by the ENCODE antibody review panel on October 17, 2016
Submitted by
Jessika Adrian
Lab
Michael Snyder, Stanford
EP300 (Mus musculus)
Method: immunoblot
Attachment from submitter
not reviewed
Submitted by
Yin Shen
Lab
Bing Ren, UCSD
EP300 (Mus musculus)
Method: ChIP-seq comparison
Attachment from submitter
not reviewed
Caption
We performed ChIP-seq of P300 in mESCs with two biological replicates, and called peaks by MACS with the default parameter (P value < 1e-5). 67% out of the 16,054 distal P300 binding sites overlap with predicted enhancers by H3K4me1, compared to random expected 6.5% (P < 2.2 x e -16 (chi-square test). This result shows that the P300 antibody we use is specific.
Submitted by
Yin Shen
Lab
Bing Ren, UCSD
EP300 (Mus musculus)
liverlungmidbrainhindbrainheartkidneystomachforebrainintestinelimb
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 several tissue 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
Mark Mackiewicz
Lab
Richard Myers, HAIB
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