ENCAB000AEV

Homo sapiens

K562, HepG2, SK-N-SH, A549

characterized to standards

Homo sapiens

GM12878, H1

not characterized to standards

Method: ChIP-seq comparison

compliant

Caption: This validation relies on the use of antibodies to chromatin regulators in K562 cells, and demonstrates highly similar patterns of enrichment are obtained with each antibody. The first track shown used an antibody to CBX2 (PchAb 165, ENCAB000AEV), and the second track shown used an antibody to CBX8 (PchAb 159, ENCAB000AEX). The third track shows data of a functionally related histone modification (H3K27me3) previously characterized to Encode standards (PchAb 67-V, ENCAB000ASB) – the use of this data is meant to act as a reference for the location in the genome rather than to quantitate to the track-track comparison between CBX2 and CBX8.
Submitted by: Nina Farrell
Lab: Bradley Bernstein, Broad
Grant: U54HG006991
Download: CBX2 PchAb 165 SAV.docx.pdf
Documents: encode:TF_Antibody_Characterization_ENCODE3_May2016.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 (56kDa) for A549 and outside of the expected size for both A549 and H1.
Submitted by: Noam Shoresh
Lab: Bradley Bernstein, Broad
Grant: U54HG006991
Download: CBX2_Bethyl_A302-524A_A302-524A-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 with an Invitrogen 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. a band was detected at expected size in all cell types (~70kDa).
Reviewer comment: Only SKNSH is compliant. MW is actually 56kDa not 70. need to fix arrow and expected size
Submitted by: Noam Shoresh
Lab: Bradley Bernstein, Broad
Grant: U54HG006991
Download: CBX2_Bethyl_A302-524A_A302-524A-1_WB.png
Documents: encode:Antibody_Characterization_ENCODE3_February2014.pdf
encode:Antibody_Characterization_ENCODE3_August2015.pdf

Method: ChIP-string comparison

not reviewed

K562

Method: immunoblot

compliant