Anti-PI 3 Kinase p85 beta antibody [EPR18416]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling PI 3 Kinase p85 beta using ab180967 at 1/100 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Cytoplasm staining on epithelial cells of Human colon was observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (ab180967) at 1/2000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 81 kDa

Observed band size: 82 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (ab180967) at 1/2000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

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

Lane 3:

HEK293 (Human epithelial cells from embryonic kidney) 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 1/1000 dilution

Predicted band size: 81 kDa

Observed band size: 82 kDa

false

Exposure time: 30s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling PI 3 Kinase p85 beta with ab180967 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and weakly cytoplasmic staining on Jurkat cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows :
1. ab180967 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

Immunohistochemical analysis of paraffin-embedded Human colonic adenocarcinoma tissue labeling PI 3 Kinase p85 beta using ab180967 at 1/100 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Nucleus and cytoplasm staining on tumor cells of colonic adenocarcinoma was observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling PI 3 Kinase p85 beta using ab180967 at 1/100 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Nucleus and cytoplasm staining on spermatogenic cell of rat testis was observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling PI 3 Kinase p85 beta with ab180967 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and cytoplasmic staining on HeLa cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows :
1. ab180967 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

PI 3 Kinase p85 beta was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab180967 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab180967 at 1/10000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : HeLa whole cell lysate10 μg (Input).

Lane 2 : ab180967 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab180967 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Immunoprecipitation - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (ab180967)

Predicted band size: 81 kDa

false

• WB

Lab

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : PIK3R2 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lanes 1 - 3 : Merged signal (red and green). Green - ab180967 observed at 85 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab180967 was shown to specifically react with PIK3R2 (KO) when PIK3R2 (KO) knockout samples were used. Wild-type and PIK3R2 (KO) knockout samples were subjected to SDS-PAGE. ab180967 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 2000 dilution and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (ab180967)

Predicted band size: 81 kDa

false

• WB

Lab

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

Lanes 1- 2 : Merged signal (red and green). Green - ab180967 observed at 85 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab180967 was shown to react with PI 3 Kinase p85 beta in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266799 (knockout cell lysate ab257586) was used. Wild-type HEK-293T and PIK3R2 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab180967 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 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 beta antibody [EPR18416] (ab180967) at 1/2000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

PIK3R2 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human PIK3R2 (PI 3 Kinase p85 beta) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-pik3r2-pi-3-kinase-p85-beta-knockout-hek-293t-cell-line-ab266799'>ab266799</a>)

Predicted band size: 56 kDa,81 kDa

Observed band size: 56 kDa,85 kDa

false

• WB

Supplier Data

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (AB180967)

5% NFDM/TBST : Blocking and diluting buffer.

All lanes:

Western blot - Anti-PI 3 Kinase p85 beta antibody [EPR18416] (ab180967) at 1/2000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

Rat spleen lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 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 1/1000 dilution

Predicted band size: 81 kDa

Observed band size: 82 kDa

false

Exposure time: 30s