Anti-WIPI2抗体[2A2]

• WB

Lab

Western blot - Anti-WIPI2 antibody [2A2] (AB105459)

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.

All lanes:

Western blot - Anti-WIPI2 antibody [2A2] (ab105459) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Wipi2 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Saos2 whole cell lysate at 20 µg

Lane 4:

CACO2 whole cell lysate at 20 µg

Predicted band size: 49 kDa

Observed band size: 49 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-WIPI2 antibody [2A2] (AB105459)

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

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-WIPI2 antibody [2A2] (AB105459)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-WIPI2 antibody [2A2] (AB105459)

ab105459 staining WIPI2 in HeLa Chloroquine treated cells. The cells were fixed with 100% methanol 5 min), permeabilized with 0.1% PBS-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 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L Alexa Fluor^® 488) preadsorbed at 1/1000 dilution shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L Alexa Fluor^® 594) at 1/1000 dilution shown in pseudocolour magenta). Nuclear DNA was labelled with DAPI shown in blue).

Image was acquired with a high-content analyser Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Supplier Data

Western blot - Anti-WIPI2 antibody [2A2] (AB105459)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the higher MW band at approximately 250kDa is unknown In Western blot, anti-WIPI2 antibody (ab105459) staining at 1/1000 dilution.Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-WIPI2 (phospho S413) antibody [EPR27037-149] (<a href='/products/primary-antibodies/wipi2-phospho-s413-antibody-epr27037-149-ab310325'>ab310325</a>) at 1/1000 dilution

Lane 1:

Untreated wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 20 µg

Lane 2:

HAP1 treated with 200 nM TPA for 30 min, whole cell lysate at 20 µg

Lane 3:

WIPI2 knockout HAP1 whole cell lysate

Lane 4:

WIPI2 knockout HAP1 treated with 200 nM TPA for 30 min, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 50 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-WIPI2 antibody [2A2] (AB105459)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the higher MW band at approximately 250kDa is unknown. In Western blot, anti-WIPI2 antibody (ab105459) staining at 1/1000 dilution.Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-WIPI2 (phospho S413) antibody [EPR27037-149] (<a href='/products/primary-antibodies/wipi2-phospho-s413-antibody-epr27037-149-ab310325'>ab310325</a>) at 1/1000 dilution

Lane 1:

Untreated KARPAS-299 (human T cell lymphoma cell) starved overnight whole cell lysate (untreated membrane) at 20 µg

Lane 2:

KARPAS-299 starved overnight, then treated with 200nM TPA for 30min whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated KARPAS-299 starved overnight whole cell lysate (phosphatase treated membrane) at 20 µg

Lane 4:

KARPAS-299 starved overnight, then treated with 200nM TPA for 30min whole cell lysate (phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 50 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-WIPI2 antibody [2A2] (AB105459)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the higher MW band at approximately 250kDa is unknown In Western blot, anti-WIPI2 antibody (ab105459) staining at 1/1000 dilution.Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-WIPI2 (phospho S413) antibody [EPR27037-149] (<a href='/products/primary-antibodies/wipi2-phospho-s413-antibody-epr27037-149-ab310325'>ab310325</a>) at 1/1000 dilution

Lane 1:

Untreated Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate

Lane 2:

Raji starved overnight, then treated with 200nM TPA for 30min whole cell lysate

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 50 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-WIPI2 antibody [2A2] (AB105459)

Abcam recommends using milk as the blocking agent.

All lanes:

Western blot - Anti-WIPI2 antibody [2A2] (ab105459) at 1 µg/mL

Lane 1:

Human skeletal muscle tissue lysate at 10 µg

Lane 2:

Human placenta tissue lysate at 10 µg

Lane 3:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

NIH 3T3 (Mouse) Whole Cell Lysate at 10 µg

Lane 5:

Pancreas (Mouse) Tissue Lysate at 10 µg

Lane 6:

Testis (Mouse) Tissue Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 49 kDa

true

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-WIPI2 antibody [2A2] (AB105459)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The identity of the higher MW band at approximately 250kDa is unknown. In Western blot, anti-WIPI2 antibody (ab105459) staining at 1/1000 dilution.Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-WIPI2 (phospho S413) antibody [EPR27037-149] (<a href='/products/primary-antibodies/wipi2-phospho-s413-antibody-epr27037-149-ab310325'>ab310325</a>) at 1/1000 dilution

Lane 1:

Untreated MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate at 20 µg

Lane 2:

MEF treated with 5uM MG-132 for 6h whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 50 kDa

false

Exposure time: 180s