All lanes : Anti-WIPI2 antibody [2A2] (ab105459) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Wipi2 knockout HAP1 whole cell lysate
Lane 3 : Saos2 whole cell lysate
Lane 4 : CACO2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 49 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab105459 observed at 49 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab105459 was shown to recognize WIPI2 in wild-type HAP1 cells as signal was lost at the expected MW in Wipi2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Wipi2 knockout samples were subjected to SDS-PAGE. Ab105459 and ab181602 (Rabbit 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab10549).

ab105459 staining WIPI2 in HeLa cells. The cells were treated with 50uM chloroquine for 24h and then fixed with 4% paraformaldehyde (10 min), 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 ab105459 at 1ugml then detected with an Alexa Fluor^® 488 goat anti-rabbit secondary antibody (ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab10549).

IHC image of WIPI2 staining in a section of formalin-fixed paraffin-embedded normal human skeletal muscle* performed on a Leica BOND^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab105459, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab10549).

Overlay histogram showing HeLa cells stained with ab105459 (red line). The cells were fixed with 80% methanol (5 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 (ab105459, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 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 and sodium azide (ab10549).

All lanes : Anti-WIPI2 antibody [2A2] (ab105459) at 1 µg/ml

Lane 1 : Human skeletal muscle tissue lysate
Lane 2 : Human placenta tissue lysate
Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 4 : NIH 3T3 (Mouse) Whole Cell Lysate
Lane 5 : Pancreas (Mouse) Tissue Lysate
Lane 6 : Testis (Mouse) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/5000 dilution

Predicted band size: 49 kDa

Abcam recommends using milk as the blocking agent.

This data was developed using the same antibody clone in a different buffer formulation containing PBS and sodium azide (ab10549).