Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free

9 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling HNF-4-alpha with purified Anti-HNF-4-alpha antibody [EPR3648] ab92378 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HNF-4-alpha antibody [EPR3648] ab92378).

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  Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling HNF-4 with purified Anti-HNF-4-alpha antibody [EPR3648] ab92378 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/500) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

  Control 1: primary antibody (1/100) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

  Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HNF-4-alpha antibody [EPR3648] ab92378).

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  Immunocytochemistry/ Immunofluorescence - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  Immunocytochemistry/Immunfluorescence analysis of HepG2 cells labelling HNF-4-alpha with unpurified Anti-HNF-4-alpha antibody [EPR3648] ab92378 at a 1/100 dilution.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HNF-4-alpha antibody [EPR3648] ab92378).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  This IHC data was generated using the same anti-HNF4 alpha antibody clone, EPR3648, in a different buffer formulation (cat# Anti-HNF-4-alpha antibody [EPR3648] ab92378).
  

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue (A) and human kidney tissue (B) labelling HNF-4-aplha with unpurified Anti-HNF-4-alpha antibody [EPR3648] ab92378 at a 1/100 dilution. Detection: DAB staining.

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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  Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  Intracellular Flow Cytometry analysis of HepG2 cells labelling HNF-4 with purified Anti-HNF-4-alpha antibody [EPR3648] ab92378 at 1/70 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
  

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HNF-4-alpha antibody [EPR3648] ab92378).

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  Flow Cytometry (Intracellular) - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  Overlay histogram showing HepG2 cells stained with unpurified Anti-HNF-4-alpha antibody [EPR3648] ab92378 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (unpurified Anti-HNF-4-alpha antibody [EPR3648] ab92378, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
  

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HNF-4-alpha antibody [EPR3648] ab92378).

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  Western blot - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  This data was developed using the same antibody clone in a different buffer formulation (abAB92378).

  Western blot: Anti-HNF4A antibody [EPR3648] (Anti-HNF-4-alpha antibody [EPR3648] ab92378) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-HNF-4-alpha antibody [EPR3648] ab92378 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.

  All lanes: Western blot - Anti-HNF-4-alpha antibody [EPR3648] (Anti-HNF-4-alpha antibody [EPR3648] ab92378) at 1/2000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: HNF4A knockout A549 cell lysate at 20 µg

  Lane 3: HepG2 cell lysate at 20 µg

  Lane 4: HEK-293 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: 50 kDa, 55 kDa

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  ChIC/CUT&RUN sequencing - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  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 Anti-HNF-4-alpha antibody [EPR3648] ab92378 [EPR3648]. 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.
  The ChIP data was conducted on chromatin prepared from HepG2 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HepG2 cells and 8 µg of Anti-HNF-4-alpha antibody [EPR3648] ab92378. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
  
  Additional screenshots of mapped reads can be downloaded here.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.
  
  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HNF-4-alpha antibody [EPR3648] ab92378).

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  ChIP-sequencing - Anti-HNF-4-alpha antibody [EPR3648] - BSA and Azide free (ab227997)

  Chromatin was prepared from HepG2 (Human liver hepatocellular carcinoma cell line) cells. ChIP was performed with 10^7 HepG2 cells and 8 µg of Anti-HNF-4-alpha antibody [EPR3648] ab92378 [EPR3648]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-HNF-4-alpha antibody [EPR3648] ab92378).