Chromatin was prepared from HepG2 (human hepatocellular carcinoma epithelial cell) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 5 µg of ab200142 (blue), and 20 µl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach).

ChIP was performed according to the literature (PMID: 18850323).

All lanes : Anti-HNF-4-alpha antibody [EPR19265-130] - ChIP Grade (ab200142) at 1/2000 dilution

Lane 1 : Human colon lysate
Lane 2 : Human fetal liver lysate
Lane 3 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : Caco-2 (human colorectal adenocarcinoma cell line) whole cell lysate
Lane 5 : SW480 (human colorectal adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/2000 dilution

Developed using the ECL technique.

Predicted band size: 53 kDa
Observed band size: 53 kDa

 

Exposure times: Lanes 1 and 2: 3 minutes; Lanes 3 and 4: 15 seconds; Lane 5: 3 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (human liver hepatocellular carcinoma cell line) cells labeling HNF-4-alpha with ab200142 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Confocal image showing nuclear staining on HepG2 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HepG2 (human liver hepatocellular carcinoma cell line) cell line labeling HNF-4-alpha with ab200142 at 1/600 (red) compared with Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

HNF-4-alpha was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab200142 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab200142 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: HepG2 (human hepatocellular carcinoma epithelial cell) 10 μg (Input).
Lane 2: ab200142 IP in HepG2 whole cell lysate (+).
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200142 in HepG2 whole cell lysate (-).
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SW480 (human colorectal adenocarcinoma cell line) cells labeling HNF-4-alpha with ab200142 at 1/100 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution (green). Confocal image showing nuclear staining on SW480 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.