Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-3 (human prostate adenocarcinoma epithelial cell) cells labelling FOXA2 with ab256493 at 1/100 (5.8 ug/ml) dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in PC-3 cell line. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (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 ab256493 anti- FOXA2 ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Immunohistochemical analysis of paraffin-embedded Human prostate neuroendocrine carcinoma tissue labeling FOXA2 with ab256493 at 1/1000 dilution (0.58ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in human prostate neuroendocrine carcinoma (PMID : 18156212;28621319). Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized LNCaP (Human prostate carcinoma epithelial cell, Left) / PC-3 (Human prostate adenocarcinoma epithelial cell, Right) cells labelling FOXA2 with ab256493 at 1/600 (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 : LNCaP (28621319, 16001449).

• IP

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Immunoprecipitation - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

FOXA2 was immunoprecipitated from 0.35 mg PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate 10ug with ab256493 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256493 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2 : ab256493 IP in PC-3 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab256493 in PC-3 whole cell lysate

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

Exposure time : 15 seconds.

All lanes:

Immunoprecipitation - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (ab256493)

Predicted band size: 48 kDa

Observed band size: 48 kDa

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• ChIP

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ChIP - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Chromatin was prepared from HepG2 cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab256493 (red), or 5 μg of rabbit normal IgG ab172730 (gray) and 20 μl of Protein A/G Sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

Primers and probes are from paper PMCID : PMC6515469.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling FOXA2 with ab256493 at 1/1000 dilution (0.58ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in rat colon (PMID : 15567715). Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling FOXA2 with ab256493 at 1/1000 dilution (0.58ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining in mouse liver (PMID : 15567715). Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling FOXA2 with ab256493 at 1/1000 dilution (0.58ug/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Negative control : No staining in mouse testis (PMID : 15567715). Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• WB

Lab

Western blot - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Blocking and Dilution Buffer and concentration : 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID : 16001449)

This blot was developed using a higher sensitivity ECL substrate for 3 minutes.

All lanes:

Western blot - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (ab256493) at 1/1000 dilution

All lanes:

SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 48 kDa

Observed band size: 48 kDa

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• WB

Lab

Western blot - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Western blot : Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade ab256493 staining at 1/1000 dilution, shown in green; anti-GST antibody [3G10/1B3] ab92 staining at 1 g/mL, shown in magenta. A band was observed at 78 kDa. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (ab256493) at 1/1000 dilution

Lane 1:

FOXA1 Recombinant Protein (GST Tag) at 0.1 µg

Lane 2:

FOXA2 Recombinant Protein (GST Tag) at 0.1 µg

Lane 3:

FOXA3 Recombinant Protein (GST Tag) at 0.1 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 78 kDa

Observed band size: 78 kDa

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• WB

Lab

Western blot - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (AB256493)

Blocking and Dilution Buffer and concentration : 5% NFDM/TBST.

Negative control : LNCaP (PMID : 28621319,16001449).

The expression profile is consistent with what has been described in the literature (PMID : 28621319, 16001449)

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (ab256493) at 1/1000 dilution

Lane 1:

Human colon cancer tissue lysate at 20 µg

Lane 2:

PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

LNCaP (human prostate carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lanes 2 - 3:

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

Predicted band size: 48 kDa

Observed band size: 48 kDa

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