All lanes : Anti-CDKN2A/p16INK4a antibody [EP435Y-129R] - BSA and Azide free (ab219723) at 1/10000 dilution

Lane 1 : HEK-293 cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/20000 dilution

Predicted band size: 17 kDa


Exposure time: 3 seconds

Clone EP435Y-129R (ab219723) has been successfully conjugated by Abcam. This image was generated using Anti-CDKN2A/p16INK4a antibody [EP435Y-129R] (Alexa Fluor® 647). Please refer to ab199819 for protocol details.

ab199819 staining CDKN2A/p16INK4a in HeLa cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab199819 at 1/200 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 2µg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone EP435Y-129R (ab219723) has been successfully conjugated by Abcam. This image was generated using Anti-CDKN2A/p16INK4a antibody [EP435Y-129R] (Alexa Fluor® 488). Please refer to ab199756 for protocol details.

ab199756 staining CDKN2A/p16INK4a in HeLa cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab199756 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293 (Human embryonic kidney epithelial cell) cells labelling CDKN2A with ab219723 at 1:100 dilution (1μg)/ 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. Gated on viable cells.

CDKN2A/p16INK4a was immunoprecipitated from 0.35 mg HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10μg with ab219723 at 1:50 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab219723 1:1000 dilution (2 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.

Lane 1: HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10μg
Lane 2: ab219723 IP in HEK-293 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab219723 in HEK-293 whole cell lysate.

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

Exposure time: 3 seconds

Overlay histogram showing HEK293 cells stained with ab81278 (red line). The cells were fixed with 4% paraformaldehyde (10 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 followed by the antibody (ab81278, 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 HEK293 cells fixed with 80% methanol (5 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 (ab81278).

This ICC data was generated using the same anti-CDKN2A antibody clone, EP435Y-129R, in a different buffer formulation (cat# ab81278).

Immunofluorescent staining of HeLa cells using 1/100 ab81278