Overview

  • Product name

    Phospho-PI 3 kinase p85 + Total In-Cell ELISA Kit
    See all PI 3 Kinase p85 alpha kits
  • Detection method

    Colorimetric
  • Sample type

    Adherent cells, Suspension cells
  • Assay type

    Cell-based (quantitative)
  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    Phospho-PI 3 kinase p85 + Total In-Cell ELISA Kit (ab207484) provides a simple, efficient, cell-based method to monitor proteins activated by phosphorylation. The kit is designed specifically to quantify activated (phosphorylated) phosphoinositide 3 Kinase (PI3K) p85 and/or total PI 3 Kinase p85. Cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are rapidly fixed to preserve activation-specific protein modifications. Each well is then incubated with a primary antibody that recognizes either phosphorylated PI 3 Kinase p85 or total PI 3 Kinase p85. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides an easily quantified colorimetric readout. The relative number of cells in each well is then determined using the provided Crystal Violet solution. The 96-well plate format is suitable for high-throughput screening applications.


    The Phospho-PI 3 kinase p85 + Total In-Cell ELISA Kit contains two 96-well plates and two primary antibodies. The phospho-PI 3 kinase p85 antibody recognizes peptides and proteins containing p85 PI 3-Kinase phospho-tyrosine 467 (alpha) or 199 (gamma). No cross-reactivity is observed with corresponding non-phosphorylated sequences or with other phospho-tyrosine-containing motifs. The total-PI 3 Kinase p85 antibody recognizes the total level of PI 3 Kinase p85 proteins regardless of the phosphorylation state. The kit can be used to study phosphorylated PI 3 Kinase relative to cell number or to determine PI 3 Kinase phosphorylation relative to the total PI 3 Kinase protein found in the cells. Once the phospho-PI 3 Kinase and total-PI 3 Kinase signals have been normalized for cell number, a comparison of the ratio of phosphorylated PI 3 Kinase to total PI 3 Kinase for each of the cell growth conditions can be made. The provided total-PI 3 Kinase antibody can be used as a positive control to demonstrate that the cells contain PI 3 Kinase, the kit reagents are functional and that the protocol is performed correctly. Also, because fixed cells are stable for several weeks, you can prepare many plates simultaneously and then perform the assay when desired.

  • Notes

    Phosphoinositide 3 Kinase (PI3 kinase, PI3K) is a major signaling molecule that is located downstream of growth factor receptor tyrosine kinases (RTKs). PI 3 Kinase catalyzes the production of the lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP3) at the cell membrane. PIP3 then contributes to the recruitment and activation of a wide range of downstream targets, including the serine-threonine protein kinase AKT (PKB). The PI 3 Kinase -AKT signaling pathway regulates many normal cellular processes including cell proliferation, survival, growth and motility-processes that are critical for tumorigenesis.

    PI 3 Kinase is a heterodimer with a p85 regulatory subunit and p110 catalytic subunit. The p85 subunit has two SH2 domains, which bind to phospho-tyrosine with the consensus motif (Y*XXM), where X denotes any amino acid and Y* is phosphorylated tyrosine. This motif exists in many receptor tyrosine kinases and their substrates, such as EGFR, FGFR, IRS-1, BCAP and GAB1. Activation of the PI 3 Kinase pathway occurs when the p85 subunit recognizes phosphorylated YXXM in signaling molecules. The small GTPase Ras can also recruit and activate PI 3 Kinase through direct binding to p110.

    A large number of downstream effector targets have been described for PI 3 Kinase. AKT/PKB is a downstream effector of PI 3 Kinase as well as p85/p110. ERK1/2 MAPK is also a target of the PI 3 Kinase signaling cascade. Some Rho family GTPases (Rac, Cdc42) are regulated by PI 3 Kinase in several systems. A link between PI 3 Kinase, phospholipase D activation, actin rearrangement and vesicular trafficking may also exist.

  • Tested applications

    Suitable for: In-Cell ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    1% SDS Solution 1 x 22ml 5 x 22ml
    10% Triton X-100 1 x 10ml 5 x 10ml
    10X PBS 1 x 120ml 5 x 120ml
    1X Antibody Blocking Buffer 1 x 22ml 5 x 22ml
    1X Antibody Dilution Buffer 1 x 30ml 5 x 30ml
    96-well tissue culture plate 2 units 10 units
    Anti-rabbit HRP-conjugated IgG 2 x 11µl 10 x 11µl
    Crystal Violet Solution 1 x 22ml 5 x 22ml
    Developing Solution 2 x 11ml 10 x 11ml
    Phospho-PI 3 Kinase p85 antibody 1 x 9µl 5 x 9µl
    Plate sealer 2 units 10 units
    Stop Solution 2 x 11ml 10 x 11ml
    Total-PI 3 Kinase p85 antibody 1 x 9µl 5 x 9µl
  • Research areas

  • Function

    Binds to activated (phosphorylated) protein-Tyr kinases, through its SH2 domain, and acts as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane. Necessary for the insulin-stimulated increase in glucose uptake and glycogen synthesis in insulin-sensitive tissues.
  • Tissue specificity

    Isoform 2 is expressed in skeletal muscle and brain, and at lower levels in kidney and cardiac muscle. Isoform 2 and isoform 4 are present in skeletal muscle (at protein level).
  • Sequence similarities

    Belongs to the PI3K p85 subunit family.
    Contains 1 Rho-GAP domain.
    Contains 2 SH2 domains.
    Contains 1 SH3 domain.
  • Domain

    The SH3 domain mediates the binding to CBLB, and to HIV-1 Nef.
  • Post-translational
    modifications

    Polyubiquitinated in T-cells by CBLB; which does not promote proteasomal degradation but impairs association with CD28 and CD3Z upon T-cell activation.
    Phosphorylated. Dephosphorylated by PTPRJ.
  • Information by UniProt
  • Alternative names

    • GRB1
    • p85
    • p85 alpha
    • P85A_HUMAN
    • Phosphatidylinositol 3 kinase associated p 85 alpha
    • Phosphatidylinositol 3 kinase regulatory 1
    • Phosphatidylinositol 3 kinase, regulatory subunit, polypeptide 1 (p85 alpha)
    • Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha
    • Phosphatidylinositol 3-kinase regulatory subunit alpha
    • Phosphoinositide 3 kinase, regulatory subunit 1 (alpha)
    • PI3 kinase p85
    • PI3 kinase p85 subunit alpha
    • PI3-kinase regulatory subunit alpha
    • PI3-kinase subunit p85-alpha
    • PI3K
    • PI3K p85
    • PI3K regulatory subunit alpha
    • Pik3r1
    • PtdIns 3 kinase p85 alpha
    • PtdIns-3-kinase regulatory subunit alpha
    • PtdIns-3-kinase regulatory subunit p85-alpha
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab207484 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent concentration.

Images

  • NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 50 ng/mL of PDGF for 5 minutes and fixed. Total and phospho PI 3 kinase p85 were each assayed in triplicate using the phospho and total PI 3 Kinase p85 antibodies included in the PI3 Kinase Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Protocols

References

ab207484 has not yet been referenced specifically in any publications.

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