All lanes : Anti-FOXA1 antibody [EPR10881] - ChIP Grade (ab170933) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : FOXA1 knockout HAP1 whole cell lysate
Lane 3 : MCF7 whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 49 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab170933 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.

Unpurified ab170933 was shown to specifically react with FOXA1 in wild-type HAP1 cells as signal was lost in FOXA1 knockout cells. Wild-type and FOXA1 knockout samples were subjected to SDS-PAGE. Ab170933 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging. 

Chromatin was prepared from LNCaP cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab170933 (red), and 20µl of protein A/G sepharose beads slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

All lanes : Anti-FOXA1 antibody [EPR10881] - ChIP Grade (ab170933) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : FOXA1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 49 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab170933 observed at 52 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab170933 was shown to react with FOXA1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261823 (knockout cell lysate ab256920) was used. Wild-type HeLa and FOXA1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab170933 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-FOXA1 antibody [EPR10881] - ChIP Grade (ab170933) at 1.1 µg/ml (purified)

Lane 1 : SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Mouse lung lysates
Lane 3 : Rat lung lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 49 kDa
Observed band size: 49 kDa

Blocking and diluting buffer: 5% NFDM/TBST.

Immunocytochemistry/ Immunofluorescence analysis of PC-3 (Human prostate adenocarcinoma epithelial cell) cells labeling FOXA1 with Purified ab170933 at 1:100 dilution (11 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor®594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor®488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat pancreas tissue sections labeling FOXA1 with Purified ab170933 at 1:1000 dilution (1.13 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0) ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse liver tissue sections labeling FOXA1 with Purified ab170933 at 1:1000 dilution (1.13 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0) ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast cancer tissue sections labeling FOXA1 with Purified ab170933 at 1:1000 dilution (1.13 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0) ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylinwas used as a counterstain.

Immunofluorescence analysis of HepG2 cells labeling FOXA1 with unpurified ab170933 at 1/100 dilution.

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling FOXA1 with unpurified ab170933 at 1/100 dilution. Heat mediated antigen retrieval was performed using citrate buffer pH 6.0 before commencing with IHC staining protocol.

Flow Cytometry analysis of HepG2 (human hepatocellular carcinoma) cells labeling FOXA1 with purified ab170933 at 1/200 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Immunohistochemical analysis of paraffin-embedded Human prostate tissue labeling FOXA1 with unpurified ab170933 at 1/100 dilution. Heat mediated antigen retrieval was performed using citrate buffer pH 6.0 before commencing with IHC staining protocol.