重组Anti-HNF-4-alpha抗体[EPR19265-130] - ChIP Grade (ab200142)

Western blot: Anti-HNF4A antibody [EPR19265-130] (ab200142) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab200142 was shown to bind specifically to HNF4A. A band was observed at 50-55 kDa in wild-type A549 cell lysates with no signal observed at this size in HNF4A knockout cell line. To generate this image, wild-type and HNF4A knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HepG2 (Human liver hepatocellular carcinoma cell line) cells and 5 µg of ab200142 [EPR19265-130]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

Additional screenshots of mapped reads can be downloaded here.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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).

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.

Intracellular flow cytometric analysis of4% 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.