Anti-FOXA2 antibody [EPR22919-71] - ChIP Grade (ab256493) at 1/1000 dilution + SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 20 µg

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

Predicted band size: 48 kDa
Observed band size: 48 kDa

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

Lane 1 : Human colon cancer tissue lysate
Lane 2 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : LNCaP (human prostate carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48 kDa
Observed band size: 48 kDa

 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.

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.

 

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).

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).

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).

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).

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.

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).

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.

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol