Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling PI 3 Kinase catalytic subunit alpha/PIK3CA with unpurified ab40776 at a dilution of 1/100.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

ab40776 staining PI 3 Kinase catalytic subunit alpha/PIK3CA in the human cell line Jurkat (human acute T cell leukemia) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permiabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/40. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells labelling PI 3 Kinase catalytic subunit alpha/PIK3CA with purified ab40776 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• IP

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Immunoprecipitation - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

ab40776 (purified) at a dilution of 1/20 immunoprecipitating PI 3 Kinase catalytic subunit alpha/PIK3CA in Jurkat whole cell lysate.

Lane 1 (input) : Jurkat whole cell lysate (10μg)

Lane 2 (+) : ab40776 + Jurkat whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab40776 in Jurkat whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/1000).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (ab40776)

Predicted band size: 124 kDa

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (ab40776) at 1/5000 dilution

Lane 1:

Jurkat whole cell lysate at 20 µg

Lane 2:

MCF-7 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 124 kDa

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : PI 3 Kinase catalytic subunit alpha/PIK3CA knockout HAP1 cell lysate (20 μg)
Lane 3 : Jurkat cell lysate (20 μg)
Lane 4 : MCF7 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab40776 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab40776 was shown to recognize PI 3 Kinase catalytic subunit alpha/PIK3CA when PI 3 Kinase catalytic subunit alpha/PIK3CA knockout samples were used, along with additional cross-reactive bands. Wild-type and PI 3 Kinase catalytic subunit alpha/PIK3CA knockout samples were subjected to SDS-PAGE. ab40776 and ab8245 (loading control to GAPDH) were diluted to 1/1000 and 1/10000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (ab40776)

Predicted band size: 124 kDa

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• WB

Lab

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

Lanes 1 - 3 : Merged signal (red and green). Green - ab40776 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab40776 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at a 1 : 10000 dilution for 1hr at room temperature and then imaged.

All lanes:

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (ab40776) at 1/1000 dilution

Lane 1:

Jurkat Whole Cell Lysate at 20 µg

Lane 2:

Mouse Brain Tissue Lysate at 20 µg

Lane 3:

Rat Brain Tissue Lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 124 kDa

Observed band size: 120 kDa

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• WB

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Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

All lanes:

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (ab40776) at 1/5000 dilution

All lanes:

Jurkat cell lysate at 10 µg

Predicted band size: 124 kDa

Observed band size: 110 kDa

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• WB

Lab

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

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

ab40776 may detect non-specific signals due to sample batch variance.

All lanes:

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (ab40776) at 1/1000 dilution

Lane 1:

Batch 1 MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Batch 2 MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Batch 3 MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 110 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (AB40776)

Blocking and dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (ab40776) at 1/5000 dilution

Lane 1:

Raw264.7 whole cell lysate at 20 µg

Lane 2:

NIH/3T3 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 124 kDa

Observed band size: 110 kDa

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