1. Antibodies
  2. Homo sapiens
  3. MBD2

ENCAB000BQP

Antibody against Homo sapiens MBD2

Homo sapiens
K562
characterized to standards
Homo sapiens
GM12878, MCF-7
characterized to standards with exemption
Status
released
Source (vendor)
Active Motif
Product ID
39548
Lot ID
33108001
Characterized targets
MBD2 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
affinity
Isotype
IgG
Antigen description
This MBD2 antibody was raised against a synthetic peptide corresponding to human MBD2
External resources

Characterizations

MBD2 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
Attachment from submitters
compliant
Caption
MCF7 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 39548). 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 MCF7: 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, MBD2, was identified as the 6th ranked enriched protein and the 1st ranked transcription factor based on IP-Mass Spectrometry.
Reviewer comment
Mass spec in the MCF-7 cell line
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
 MBD2-M-WL.png
MBD2 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
Attachment from submitters
compliant
Caption
Analysis of gel fragment 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, MBD2, was identified as the 13th ranked enriched protein and the 6th ranked transcription factor based on IP-Mass Spectrometry.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
MBD2 (Homo sapiens)
K562GM12878MCF-7
Method: immunoblot
Attachment from submitter
Attachment from submitters
exempt from standards
Caption
Whole cell lysates of K562, HepG2, and GM12878 were immunoprecipitated using the primary antibody (Sigma; WH0006932M1). 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. Two bands were detected at ~35 and 50 kDa.
Submitter comment
--
Reviewer comment
The lane was not run out in full, so no clear bands are visible. Rescued by mass spectrometry analysis
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
MBD2 (Homo sapiens)
GM12878
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
not submitted for review by lab
Caption
GM12878 whole cell lysate was immunoprecipitated using the primary antibody (Active Motif; 39548). 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.
Submitter comment
Gel for mass spec characterization. Expected size ~43 kDa
Reviewer comment
See comment- Aditi
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
MBD2 (Homo sapiens)
K562GM12878
Method: immunoblot
Attachment from submitter
Attachment from submitters
compliant
Caption
Whole cell lysates of K562 and GM12878 were immunoprecipitated using the primary antibody (Active Motif; 39548). 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. One band was detected at ~47 kDa.
Submitter comment
Band is not 50% of signal, rescued by mass spec. Expected size not given, but should be around 43 kDa according to uniprot
Reviewer comment
See comment- Aditi
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB
Download
 MBD2_IP_WB.png