ENCAB000AGM

Homo sapiens

at least one cell type or tissue

awaiting characterization

Method: ChIP-seq comparison

not reviewed

Caption: Validation 2: Immunoprecipitation with multiple antibodies against different parts of the target protein ENCODE data standards allow for secondary validation of antibodies by performing ChIP with multiple antibodies against different parts of the target protein. A statistically significant overlap of targets constitutes validation. FOXA1 (Q-6) sc-101058 is a mouse monoclonal antibody raised against recombinant FOXA1 of human origin. A second antibody used in ChIP-seq experiments on FOXA1 in our lab is FOXA1 (C-20) sc-6553, a goat polyclonal antibody with epitope mapping at the C-terminus of FOXA1 of human origin. Irreproducible Discovery Rate (IDR) analysis results for two ChIP-seq experiments using these two antibodies, each in HepG2, are as follows: At IDR 0.01: 14930 peaks are significant At IDR 0.05: 18842 peaks are significant At IDR 0.1: 21519 peaks are significant These results indicate significant overlap between the two ChIP-seq libraries.
Submitted by: Richard Myers
Lab: Richard Myers, HAIB
Grant: U54HG004576
Download: human_FOXA1_SC-101058_validation_Myers.pdf

Method: immunoprecipitation

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-101058 in nuclear extract (NE) of HepG2 and in whole cell lysate (WCL) of HepG2, K562 and HeLa; PM=protein marker. FOXA1 bands are indicated.
Submitted by: Richard Myers
Lab: Richard Myers, HAIB
Grant: U54HG004576
Download: human_FOXA1_SC-101058_validation_Myers.pdf