Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling HNF-4-alpha with ab181604 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus staining on Human liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling HNF-4-alpha with ab181604 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on epithelial cells of Human colon is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

• ChIP

Supplier Data

ChIP - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Chromatin was prepared from HepG2 (Human liver hepatocellular carcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab181604 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

ABCC6_NC1 is negative control

• IP

Supplier Data

Immunoprecipitation - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

HNF-4 alpha was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma) whole cell extract with ab181604 at 1/70 dilution. Western blot was performed from the immunoprecipitate using ab181604 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : HepG2 whole cell extract 10 μg (Input).

Lane 2 : ab181604 IP in HepG2 whole cell extract.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab181604 in HepG2 whole cell extract.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (ab181604)

Predicted band size: 53 kDa

Observed band size: 53 kDa

false

Exposure time: 5s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling HNF-4-alpha with ab181604 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on mouse liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling HNF-4-alpha with ab181604 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on epithelial cells of rat colon is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

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

• WB

Lab

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour before being incubated with ab181604 (rabbit monoclonal [EPR16885] to HNF-4-alpha; dilution 1 : 10000) and loading control ab18058 (mouse monoclonal [SPM227] to Vinculin; dilution 1 : 10000) at 4°C overnight. Antibody binding was detected with ab216773 (Goat anti-Rabbit IgG H&L (IRDye® 800CW); green; dilution 1 : 20000) and ab216776 (Goat anti-Mouse IgG H&L (IRDye® 680RD), red; dilution 1 : 20000) for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (ab181604) at 1/10000 dilution

Lane 1:

HepG2 whole cell lysate at 20 µg

Lane 2:

HEK293 whole cell lysate at 20 µg

Lane 3:

Mouse liver lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 53 kDa

false

• WB

Supplier Data

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (ab181604) at 1/1000 dilution

All lanes:

SW480 (Human colon adenocarcinoma cell line) 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: 53 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (ab181604) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human colon lysate at 10 µg

Lane 3:

Human fetal kidney 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

Observed band size: 53 kDa

false

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (ab181604) 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: 53 kDa

false

• WB

Supplier Data

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

This antibody can recognize 2 isoforms in mouse and rat. The predicted MW are 53KDa and 52 KDa.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (ab181604) at 1/1000 dilution

Lane 1:

Mouse liver lysate at 10 µg

Lane 2:

Rat liver lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 53 kDa

false

Exposure time: 15s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HepG2 (Human liver hepatocellular carcinoma cell line) cells and 5 µg of ab181604 [EPR16885]. 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.

• WB

Supplier Data

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (AB181604)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade (ab181604) at 1/1000 dilution

All lanes:

Full length mouse HNF-4-gamma recombinant protein at 10 µg

Secondary

All lanes:

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

Predicted band size: 45 kDa,53 kDa

Observed band size: 38 kDa,39 kDa

false

Exposure time: 3min