ENCAB000BOT

Antibody against Homo sapiens EED

Homo sapiens
GM12878, K562
characterized to standards with exemption
Status
released
Source (vendor)
Active Motif
Product ID
61203
Lot ID
31511001
Characterized targets
EED (Homo sapiens)
Host
mouse
Clonality
monoclonal
Purification
Protein G
Isotype
IgG2a
Antigen description
This EED antibody was raised against full-length recombinant human EED protein.
External resources

Characterizations

EED (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61203). 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 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, EED, was identified as the 2nd enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
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EED (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61203). 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 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, EED, was identified as the 1st enriched protein and the 1st transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
EED (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
not compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 61203). 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 3 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, EED, was not identified based on IP-Mass Spectrometry.
Reviewer comment
Target of interest is not within the top 20 of the list by fold enrichment
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
EED (Homo sapiens)
K562GM12878
Method: immunoprecipitation
Attachment from submitter
not compliant
Caption
An immunoprecipitation followed by Western blot was performed with Active Motif 61203 (lot 31511001) antibody against EED in K562 and GM12878 whole cell lysate along with a control IP with normal IgG from the same host species as the test antibody (mouse, Santa Cruz sc-2025). The expected size of EED is 57 kDa. Bands of ~53, 65, and 210 kDa were detected in the test IP and not in the control IP. The experiment was repeated and these bands were analyzed by mass spec in the secondary validation.
Submitter comment
non specific bands
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB
EED (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
not reviewed
Submitter comment
Gel for mass spectrometry characterization
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
EED (Homo sapiens)
K562GM12878
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Whole cell lysates of K562 and GM12878 were immunoprecipitated using the primary antibody (ActiveMotif; 61203). 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. Three bands were detected at ~56, 64, and 210 kDa. Expected size ~50 kDa.
Submitter comment
--
Reviewer comment
Band is not 50% of overall signal, found in the control lanes.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
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