ENCAB000AHI

Homo sapiens

K562, HepG2, GM12878, SK-N-SH, A549, H1

characterized to standards

Homo sapiens

any cell type or tissue

partially characterized

Method: ChIP-seq comparison

compliant

Caption: This validation relies on the use of antibodies to different members of a known complex, in HepG2 cells, and the demonstration that highly similar patterns of enrichment are obtained with each antibody. The first track illustrates the antibody being validated (HDAC2, PchAb 317), the second track illustrates an antibody to CHD4 (Broad alias PchAb 1222), and the third track illustrates an antibody to KDM1A (Broad alias PchAb 158). Thus, this illustrates the high similarity between three members of the NuRD complex. Overall correlation scores: HDAC2 vs. CHD4: 0.8539; HDAC2 vs. KDM1A: 0.8566
Submitted by: Kathrina Onate
Lab: Bradley Bernstein, Broad
Grant: U54HG006991
Download: HDAC2 Bethyl A300-705A lot 1 SAV.pdf
Documents: encode:Histone_Modification_Antibody_Characterization_ENCODE3_Oct2016.pdf

Method: ChIP-seq comparison

not compliant

Caption: ChIP-seq was performed using two closely related cell types, and using different antibodies that were raised against different regions of the same protein, and highly similar results were obtained. This is suggestive that the enrichment obtained using ChIP is due to a specific interaction between the antibody and the intended cellular protein.
Reviewer comment: Failed due to additional information needed (needs to be compared to an eligible antibody lot, needs to list ENCAB of lot, ENCSR of data used in comparison, IDR metrics need to be reported)
Submitted by: Noam Shoresh
Lab: Bradley Bernstein, Broad
Grant: U54HG006991
Download: HDAC2_Bethyl_A300-705A_A300-705A-1_chip_compare.png
Documents: encode:Antibody_Characterization_ENCODE3_August2015.pdf

A549H1

Method: immunoblot

compliant

Caption: Nuclear lysates from A549 (10ug) and H1 (10ug) were loaded into a 4-12% Bis-Tris gel in 1X MOPS running buffer. After separation, the samples were transferred to a nitrocellulose membrane using iblot. Membrane was blocked for an hour in room temperature, with 5% BSA in TBS-T and blotted with primary antibody in the appropriate concentration over night at 4c. Membrane was washed and blotted with secondary HRP-conjugated antibody. Detection was made with Optiblot ECL Detect Kit (ab133406) for 2 min. Bands were detected at the expected size (60kDa).
Submitted by: Noam Shoresh
Lab: Bradley Bernstein, Broad
Grant: U54HG006991
Download: HDAC2_Bethyl_A300-705A_A300-705A-1_V2.png
Documents: encode:Antibody_Characterization_ENCODE3_August2015.pdf

K562HepG2GM12878SK-N-SH

Method: immunoblot

compliant

Caption: Nuclear lysates from K562 (10ug), HepG2 (7ug), GM12878 (7ug), SK-N-SH (6ug), were loaded into a 4-12% Bis-Tris gel in 1X MES running buffer. After separation, the samples were transferred to a nitrocellulose membrane using the iblot system. Membrane was blocked for an hour in room temperature, with 5% BSA in TBS-T and blotted with primary antibody in the appropriate concentration over night at 4c. Membrane was washed and blotted with secondary HRP-conjugated antibody. Detection was made with Optiblot ECL Detect Kit (ab133406) for 2 min. Band of expected size (60kDa) visualized representing strongest signal in the lane.
Submitted by: Noam Shoresh
Lab: Bradley Bernstein, Broad
Grant: U54HG006991
Download: HDAC2_Bethyl_A300-705A_A300-705A-1_WB_AS.png
Documents: encode:Antibody_Characterization_ENCODE3_February2014.pdf
encode:Antibody_Characterization_ENCODE3_August2015.pdf

Method: ChIP-string comparison

not reviewed

Method: immunoblot

not reviewed