Anti-HNF1 alpha antibody [EPR23054-108]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% Triton X-100 permeabilized HepG2 and HeLa cells labelling HNF1 alpha with ab272693 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HepG2 cells. 100% methanol fixation is recommended. Negative control : HeLa (PMID : 10677375). ab195889 Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling HNF1 alpha with ab272693 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human colon. The section was incubated with ab272693 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling HNF1 alpha with ab272693 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human liver. The section was incubated with ab272693 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell, Left) / HepG2 (Human hepatocellular carcinoma epithelial cell, Right) cells labelling HNF1 alpha with ab272693 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : HeLa (PMID : 10677375).

• WB

Lab

Western blot - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

Western blot : Anti-HNF1A antibody [EPR23054-108] (ab272693) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab272693 was shown to bind specifically to HNF1A. A band was observed at 75-80 kDa in wild-type A549 cell lysates with no signal observed at this size in HNF1A knockout cell line. To generate this image, wild-type and HNF1A 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.

All lanes:

Western blot - Anti-HNF1 alpha antibody [EPR23054-108] (ab272693) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

HNF1A knockout A549 cell lysate at 20 µg

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

U-2 OS cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 75 kDa,80 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

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 5µg of ab272693 [EPR23054-108]. 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

Lab

Western blot - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

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

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-HNF1 alpha antibody [EPR23054-108] (ab272693) at 1/1000 dilution

Lane 1:

Mouse liver tissue lysate at 20 µg

Lane 2:

Rat liver tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 67 kDa

Observed band size: 81 kDa

false

• WB

Lab

Western blot - Anti-HNF1 alpha antibody [EPR23054-108] (AB272693)

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

Lysates were made freshly and used in WB immediately to minimize protein degradation.

The molecular weight observed is consistent with what has been described in the literature (PMID : 31145732).

Negative control : HeLa (PMID : 10677375).

Exposure time : Lanes 1-3 : 10 seconds; Lane 4 : 3 minutes.

All lanes:

Western blot - Anti-HNF1 alpha antibody [EPR23054-108] (ab272693) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Hepa1-6 (mouse hepatoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 67 kDa

Observed band size: 81 kDa

false