Recombinant Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (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.

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.

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.

Blocking/Dilution buffer: 5% NFDM/TBST.

Human PI3K p85 alpha full length recombinant protein contain aa1-724 with a His-Tag® (Cat#ab84769). Human PI3K p85 beta full length recombinant protein contain aa1-728 with a His-Tag® (Cat#ab125568). 

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.

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what have been described in the literatures (PMID: 8921377, 12649157).

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 Fluorr^® 488) at 1/500 dilution was used as the secondary antibody. 

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time:  Lane 1 and 2: 10 seconds;  Lane 3 and 4: 3 seconds.

The molecular weight observed is consistent with what have been described in the literatures (PMID: 8921377, 12649157).

Overlay histogram showingHepG2 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). 

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-8: 3 minutes; Lane 9-12: 10 seconds.

The molecular weight observed is consistent with what have been described in the literatures (PMID: 8921377, 12649157).

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.