Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free (AB223792)

Overlay histogram showing HepG2 cells fixed in 4% PFA and stained with ab191606 at a dilution of 1/80 (red line). The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG (ab172730) was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191606).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free (AB223792)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling PI3K p85 with ab191606 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291)  at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor^®594) preadsorbed (ab150120)  at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab191606 at 1/500 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2 : ab7291  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191606).

• IP

Supplier Data

Immunoprecipitation - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free (AB223792)

PI3K p85 was immunoprecipitated from 1mg of MCF7 (Human breast adenocarcinoma cell line) whole cell lysate with ab191606 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab191606 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : MCF7 whole cell lysate, 10μg (Input).

Lane 2 : ab191606 IP in MCF7 whole cell lysate.

Lane 3 : Rabbit IgG, monoclonal - Isotype Control (ab172730) instead of ab191606 in MCF7 whole cell lysate.

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

Exposure time : 5 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191606).

All lanes:

Immunoprecipitation - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (<a href='/en-us/products/primary-antibodies/pi-3-kinase-p85-alpha-antibody-epr18702-ab191606'>ab191606</a>)

Predicted band size: 83 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free (AB223792)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 100% Methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PI3K p85 with ab191606 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291) at  1/1000 dilution and Goat Anti-Mouse IgG  (AlexaFluor^®594) preadsorbed (ab150120) at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab191606 at 1/500 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191606).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free (AB223792)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PI3K p85 with ab191606 at 1/150 dilution (red) compared with a Rabbit IgG,monoclonal - Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab191606).

• WB

Unknown

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free (AB223792)

This WB data was generated using the same anti-PI 3 Kinase p85 alpha antibody clone [EPR18702] in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (cat# ab191606).

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : PIK3R1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : Jurkat whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab191606 observed at 90 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab191606 was shown to specifically react with PIK3R1 when PIK3R1 knockout samples were used. Wild-type and PIK3R1 knockout samples were subjected to SDS-PAGE. ab191606 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (<a href='/en-us/products/primary-antibodies/pi-3-kinase-p85-alpha-antibody-epr18702-ab191606'>ab191606</a>)

Predicted band size: 83 kDa

false

• WB

Lab

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] - BSA and Azide free (AB223792)

This data was developed using the same antibody clone in a different buffer formulation (ab191606).

Lanes 1- 2 : Merged signal (red and green). Green - ab191606 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab191606 was shown to react with PI 3 Kinase p85 alpha in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265116 (knockout cell lysate ab257029) was used. Wild-type HeLa and PIK3R1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab191606 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (<a href='/en-us/products/primary-antibodies/pi-3-kinase-p85-alpha-antibody-epr18702-ab191606'>ab191606</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PIK3R1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PIK3R1 (PI 3 Kinase p85 alpha) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-pik3r1-pi-3-kinase-p85-alpha-knockout-hela-cell-line-ab265116'>ab265116</a>)

Predicted band size: 83 kDa

Observed band size: 90 kDa

false