, corresponding to C terminal amino acids 418-436 of Human WIPI2 (Isoform WIPI2b).
This antibody gave a positive signal in Human Skeletal Muscle, Human Placenta, Mouse Placenta, Mouse Testis as well as the following whole cell lysates: HeLa, and NIH3T3.
IF/ICC: HeLa cells (50mM chloroquine for 24h)
Flow Cyt: HeLa cells
IHC-P: Human skeletal muscle (normal)
This antibody clone is manufactured by Abcam. We welcome customer feedback and would appreciate any comments regarding this product and the data presented.
If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact firstname.lastname@example.org or you can find further information here.
Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
IHC image of WIPI2 staining in a section of formalin-fixed paraffin-embedded normal human skeletal muscle* performed on a Leica BONDTM 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
Western blot - Anti-WIPI2 antibody [2A2] (ab105459)
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 Observed 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.
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).
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, 1μg/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.