Anti-PI 3 Kinase p85 alpha 抗体 [EPR18702]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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).

• IP

Supplier Data

Immunoprecipitation - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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.

All lanes:

Immunoprecipitation - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

Predicted band size: 83 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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.

• WB

Lab

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

Compared with ab40755, ab191606 has higher sensitivity. We recommend ab124805 as an alternative for testing western blot.

We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results when using ab197896 in western blot.

Lane 1:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EP380Y] (<a href='/products/primary-antibodies/pi-3-kinase-p85-alpha-antibody-ep380y-ab40755'>ab40755</a>) at 1/1000 dilution

Lane 2:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

All lanes:

Jurkat (human T cell leukemia T lymphocyte) 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 1/20000 dilution

Observed band size: 85 kDa

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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).

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

Lane 1:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 4:

Raji (Human Burkitt's lymphoma cell line) 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

Predicted band size: 83 kDa

Observed band size: 46 kDa,85 kDa

false

• WB

Unknown

Western blot - 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.

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

Predicted band size: 83 kDa

false

• WB

Lab

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (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] (ab191606) 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='/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

• WB

Supplier Data

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 83 kDa

Observed band size: 46 kDa,85 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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).

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/20000 dilution

Lane 1:

Human PI3K p85 alpha full length recombinant protein at 0.01 µg

Lane 2:

Human PI3K p85 beta full length recombinant protein at 0.01 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

Predicted band size: 41 kDa,83 kDa

Observed band size: 55-60 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (AB191606)

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).

All lanes:

Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Mouse kidney lysate at 10 µg

Lane 4:

Mouse spleen lysate at 10 µg

Lane 5:

Rat brain lysate at 10 µg

Lane 6:

Rat heart lysate at 10 µg

Lane 7:

Rat kidney lysate at 10 µg

Lane 8:

Rat spleen lysate at 10 µg

Lane 9:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 10:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 11:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 12:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 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

Predicted band size: 83 kDa

Observed band size: 46 kDa,85 kDa

false