ENCAB000BOT
Antibody against Homo sapiens EED
Homo sapiens
K562, GM12878
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)
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
- Grant
- U54HG006998
- Download
- EED-1.pdf
EED (Homo sapiens)
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
- Grant
- U54HG006998
- Download
- EED-2.pdf
EED (Homo sapiens)
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
- Grant
- U54HG004576
- Download
- EED-3.pdf
EED (Homo sapiens)
K562GM12878
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
- Grant
- U54HG006998
- Download
- EED_61203_31511001_IP-Western.png
EED (Homo sapiens)
K562
not reviewed
- Submitter comment
- Gel for mass spectrometry characterization
- Submitted by
- Mark Mackiewicz
- Lab
- Richard Myers, HAIB
- Grant
- U54HG006998
- Download
- EED_IP-Commassie1.png
EED (Homo sapiens)
K562GM12878
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
- Grant
- U54HG006998
- Download
- EED_IP-WB.png