Anti-HNF1 alpha antibody [EPR23054-108]

8 product images

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  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 (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 67 kDa

  Observed band size: 81 kDa

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  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 (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/50000 dilution

  Predicted band size: 67 kDa

  Observed band size: 81 kDa

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  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.
  

  Negative control: HeLa (PMID: 10677375).

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  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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

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  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 (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 (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.

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  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 (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 (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.

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  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] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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

  Performed under reducing conditions.

  Observed band size: 75 kDa, 80 kDa

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  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 Rabbit IgG, monoclonal [EPR25A] - Isotype 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.