Anti-HNF-4-alpha antibody [EPR16786] - N-terminal

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (AB199431)

Immunohistochemical analysis of paraffin-embedded Human hepatocellular carcinoma tissue labeling HNF-4-alpha with ab199431 at 1/400 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nucleus staining on Human hepatocellular carcinoma tissue is observed. Counter stained with Hematoxylin.
Negative control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (AB199431)

Chromatin was prepared from HepG2 (Human liver hepatocellular carcinoma cell line) cells. ChIP was performed with 10^7 HepG2 cells and 8 µg of ab199431 [EPR16786]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (AB199431)

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 ab199431 [EPR16786]. 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. The ChIP data was conducted on chromatin prepared from HepG2 cells. ChIP was performed with 10^7 HepG2 cells and 8 µg of ab199431 [EPR16786]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (AB199431)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma) cells labeling HNF-4-alpha with ab199431 at 1/250 dilution followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on HepG2 cell line is observed. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows : -
-ve control 1 - ab199431 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

• WB

Supplier Data

Western blot - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (AB199431)

This antibody can recognize 3 isoforms. The predicted MW are 53KDa, 52 KDa and 47KDa, respectively.

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (ab199431) at 1/10000 dilution

Lane 1:

Mouse liver tissue lysate at 10 µg

Lane 2:

Rat liver tissue lysate at 10 µg

Lane 3:

Human fetal liver tissue lysate at 10 µg

Lane 4:

Human stomach tissue lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 53 kDa,84 kDa

Observed band size: 47 kDa,53 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (AB199431)

This antibody can recognize 3 isoforms. The predicted MW are 53KDa, 52 KDa and 47KDa, respectively.

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16786] - N-terminal (ab199431) at 1/10000 dilution

All lanes:

HepG2 (Human liver hepatocellular carcinoma) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 53 kDa

Observed band size: 47 kDa,53 kDa

false

Exposure time: 1min