Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] - BSA and Azide free (AB185927)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40776).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] - BSA and Azide free (AB185927)

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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40776).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] - BSA and Azide free (AB185927)

This ICC/IF data was generated using the same anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody clone, EP383Y, in a different buffer formulation (cat# 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

Unknown

Immunoprecipitation - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] - BSA and Azide free (AB185927)

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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40776).

All lanes:

Immunoprecipitation - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] (<a href='/en-us/products/primary-antibodies/pi-3-kinase-catalytic-subunit-alpha-pik3ca-antibody-ep383y-ab40776'>ab40776</a>)

Predicted band size: 124 kDa

Observed band size: 110 kDa

false

• WB

Lab

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] - BSA and Azide free (AB185927)

This WB data was generated using the same anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody clone, EP383Y, in a different buffer formulation (cat# 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 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] (<a href='/en-us/products/primary-antibodies/pi-3-kinase-catalytic-subunit-alpha-pik3ca-antibody-ep383y-ab40776'>ab40776</a>)

Predicted band size: 124 kDa

false

• WB

Lab

Western blot - Anti-PI 3 Kinase catalytic subunit alpha/PIK3CA antibody [EP383Y] - BSA and Azide free (AB185927)

This data was developed using ab40776, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/pi-3-kinase-catalytic-subunit-alpha-pik3ca-antibody-ep383y-ab40776'>ab40776</a>) 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