All lanes : Anti-Transcription factor AP-2-alpha antibody [EPR2688(2)] (ab108311) at 1/10000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : C6 (Rat glial tumor glial cell) whole cell lysate
Lane 3 : Mouse skin lysate

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: 48 kDa

Purified ab108311 at 1/20 dilution (0.5µg) immunoprecipitating Transcription factor AP-2-alpha in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab108311 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab108311 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/10,000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 48 kDa

Flow Cytometry analysis of JAR (Human placenta choriocarcinoma epithelial cell) cells labeling Transcription factor AP-2-alpha with Purified ab108311 at 1/20 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry analysis of JAR (Human placenta choriocarcinoma epithelial cell) cells labeling Transcription factor AP-2-alpha with Purified ab108311 at 1/50 dilution (3.4 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 dilution (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 dilution (2 µg/mL). DAPI (blue) was used as 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 breast tissue sections labeling Transcription factor AP-2-alpha with Purified ab108311 at 1/100 dilution (1.07 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse breast tissue sections labeling Transcription factor AP-2-alpha with Purified ab108311 at 1/100 dilution (1.07 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling Transcription factor AP-2-alpha with Purified ab108311 at 1/100 dilution (1.07 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-Transcription factor AP-2-alpha antibody [EPR2688(2)] (ab108311) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TFAP2A knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

Lanes 1- 2: Merged signal (red and green). Green - ab108311 observed at 48 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab108311 was shown to react with Transcription factor AP-2-alpha in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265122 (knockout cell lysate ab257736) was used. Wild-type HeLa and TFAP2A knockout HeLa cell lysates were subjected to SDS-PAGE. ab108311 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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-Transcription factor AP-2-alpha antibody [EPR2688(2)] (ab108311) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : TFAP2A (Transcription factor AP-2-alpha) knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 48 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab108311 observed at 48 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab108311 was shown to recognize Transcription factor AP-2-alpha in wild-type HAP1 cells as signal was lost at the expected MW in TFAP2A (Transcription factor AP-2-alpha) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and TFAP2A (Transcription factor AP-2-alpha) knockout samples were subjected to SDS-PAGE. Ab108311 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 1/20000 dilution for 1 hour at room temperature before imaging.