Phospho-PI 3 kinase p85 + Total In-Cell ELISA Kit (ab207484)
Key features and details
- Assay type: Cell-based (quantitative)
- Detection method: Colorimetric
- Sample type: Adherent cells, Suspension cells
- Reacts with: Mouse, Rat, Human
Overview
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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.
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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.
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses. -
Platform
Microplate
Properties
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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
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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
modificationsPolyubiquitinated 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
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Alternative names
- GRB1
- p85
- p85 alpha
see all -
Database links
- Entrez Gene: 5295 Human
- Entrez Gene: 18708 Mouse
- Entrez Gene: 25513 Rat
- Omim: 171833 Human
- SwissProt: P27986 Human
- SwissProt: P26450 Mouse
- SwissProt: Q63787 Rat
- Unigene: 132225 Human
see all
Images
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Measurement of phosphorylated and total PI3 kinase.
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).
