710400

antibody from Invitrogen Antibodies

Targeting: PIK3R3 p55

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

710400

Provider

Invitrogen Antibodies

Product name

PI3K p85/p55 Recombinant Polyclonal Antibody (6HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Antibody clone number

6HCLC

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PI3K was performed by loading 20 µg of K562 (lane1), MCF7 (lane2), HeLa (lane3), HEK-293 (lane4), NIH/3T3 (lane5) and Jurkat (lane6) cell lysates using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. PI3K was detected at ~80 kDa using PI3K Recombinant Rabbit Polyclonal Antibody (Product # 710400) at 0.5 µg-1 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of PI3 kinase p85 in whole extracts of Jurkat, NIH-3T3, HEK293, HeLa, MCF-7, and K562 lysate (lanes 1-5 respectively) using a PI3 kinase p85 Recombinant Rabbit Polyclonal Antibody (Product # 710400) at a dilution of 2 µg/mL. Tubulin was used as a loading control and detected with an Anti-tubulin antibody. Samples were detected using chemiluminescence (ECL). Results show a band at ~85kDa.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of PI3 kinase p85 in HeLa cells using a PI3 kinase p85 Recombinant Rabbit Polyclonal Antibody (Product # 710400) followed by detection using an Alexa Fluor 488-conjugated Goat anti-Rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor 594 phalloidin (red) (image C). Image D is a composite image showing cytoplasmic localization of PI3K.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescent analysis of PI3K was done on 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with PI3K Recombinant Rabbit Polyclonal Antibody (Product # 710400) at 2 µg-4 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic and membrane localization of PI3K. Panel e shows no primary antibody. The images were captured at 20X magnification.