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

• Flow Cyt (Intra)

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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 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 (ab92378).

• Flow Cyt (Intra)

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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 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 ab92378, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (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 (ab92378).

• ICC/IF

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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 ab92378 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. 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. ab7291, a mouse anti-tubulin (1/500) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, 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 (ab92378).

• ICC/IF

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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 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 (ab92378).

• ChIP-seq

Supplier Data

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 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 (ab92378).

• WB

Lab

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 (ab92378).

Western blot : Anti-HNF4A antibody [EPR3648] (ab92378) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, 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] (<a href='/en-us/products/primary-antibodies/hnf-4-alpha-antibody-epr3648-ab92378'>ab92378</a>) 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

Observed band size: 50 kDa,55 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

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 ab92378 [EPR3648]. 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. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HepG2 cells and 8 µg of 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 (ab92378).