ENCAB009VWX

Homo sapiens

any cell type or tissue

characterized to standards

K562

Method: immunoblot

compliant

Caption: We performed a WB using nuclear lysates K562 cells (10ug) as well as unmodified Histone H3 peptide. Samples were resolved by electrophoresis on 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 at room temperature, with 5% nonfat dry milk and blotted with primary antibody in TBST with 5% BSA at 1:1000 overnight at 4c. Membrane was washed and blotted with secondary HRP-conjugated antibody. Detection was made with Thermo Scientific SuperSignal West Femto Detect Kit (34094) for 1 min. Band of expected size (~17 kDa) visualized in K562 nuclear extract and cannot be detected in unmodified recombinant Histone H3. Antibody seems to be specific since no reactivity with non histone proteins or unmodified histones was detected.
Submitted by: Nina Farrell
Lab: Bradley Bernstein, Broad
Grant: UM1HG009390
Download: H3K27me3_Cell Signaling_9733_14_WB_ PchAb 1409.pdf
Documents: encode:Histone_Modification_Antibody_Characterization_ENCODE3_Oct2016.pdf

Method: dot blot assay

compliant

Caption: We performed a DB analysis using the large set of modified peptides provided in the Active Motif MODified Histone Peptide Array, catalog #13005. The only peptide recognized by the H3K27me3 antibody was the correct one with very little cross reactivity.
Submitted by: Nina Farrell
Lab: Bradley Bernstein, Broad
Grant: UM1HG009390
Download: H3K27me3_Cell Signaling_Part_9733_Lot_14_DB_PchAb 1409.pdf
Documents: encode:Histone_Modification_Antibody_Characterization_ENCODE3_Oct2016.pdf