ENCAB000ANS

Homo sapiens

at least one cell type or tissue

awaiting characterization

Method: immunoblot

not reviewed

Caption: Western Blot analysis: 30 μg nuclear extract from Ntera-2 cells was loaded onto 10% SDS PAGE, proteins were transferred onto nitro cellulose membrane and probed with rabbit anti YY1 antibody (sc1703X) at 1ug/mL in TBST+5% milk. Only one major band was detected at ~60kDa corresponding to YY1.
Submitted by: Peggy Farnham
Lab: Michael Snyder, Stanford
Grant: U54HG004558
Download: human_YY1_sc-1703_validation_Farnham.pdf

Method: motif enrichment

not reviewed

Caption: YY1 motif analysis in NTERA2 YY1 (H-414) ChIP-seq peaks. We used the YY1 (H-414) ChIP-seq data in NTERA2 cells to search for the recognized YY1 motif CGCCATnTT (shown in reverse compliment in the motif logo). Shown in the graph is the percentage of YY1 sites with a YY1 motif in ranked bins of 250 ChIP-seq peaks. As the graph shows, greater than 80% of the top ranked peaks contain the YY1 DNA-binding motif.
Submitted by: Peggy Farnham
Lab: Michael Snyder, Stanford
Grant: U54HG004558
Download: human_YY1_sc-1703_validation_Farnham.pdf
Documents: encode:TF_Antibody_Characterization_ENCODE3_May2016.pdf

Method: immunoprecipitation followed by mass spectrometry

not reviewed

Caption: IP followed by mass spectrometry: Briefly, K562 whole cell lysates were immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. Gel was stained with Coomassie Blue in order to visualize marker bands. Gel fragments corresponding to the bands indicated above in the western blot image were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. There the samples were run on an LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using MASCOT and Scaffold tandem mass spectra analysis, with probability based matching at FDR < 0.05. As per ENCODE data standards, all Scaffold results are attached (ENCODE_HAIB_YY1_sc1703_MassSpec.pdf), including common contaminants. Target protein is highlighted.
Submitted by: Richard Myers
Lab: Richard Myers, HAIB
Grant: U54HG004576
Download: human_YY1_sc-1703_validation_Myers.pdf

Method: immunoblot

not reviewed

Caption: Western blot protocol: Whole cell lysate was immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. Blotting with primary (same as that used for IP) and secondary HRP-conjugated antibodies was performed on an Invitrogen BenchPro 4100 system. Visualization was achieved using SuperSignal West Femto solution (Thermo Scientific). Results: Band of expected size visualized, representing strongest signal in the lane. Figure legend: IP-western with sc-1703 in whole cell lysates (WCL) of K562 and GM12878. YY1 band is indicated, as is heavy chain of IgG. PM=Protein Marker.
Submitted by: Richard Myers
Lab: Richard Myers, HAIB
Grant: U54HG004576
Download: human_YY1_sc-1703_validation_Myers.pdf