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Human Phosphoinositide-3-Kinase Catalytic Beta Polypeptide (PIK3Cb) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to PIK3Cb. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to PIK3Cb. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain PIK3Cb, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of PIK3Cb in the samples is then determined by comparing the O.D. of the samples to the standard curve.


For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Phosphoinositide-3-kinase (PI3K) phosphorylates phosphatidylinositol derivatives at position 3 of the inositol ring to produce 3-phosphoinositides (PubMed:15135396). Uses ATP and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3) (PubMed:15135396). PIP3 plays a key role by recruiting PH domain-containing proteins to the membrane, including AKT1 and PDPK1, activating signaling cascades involved in cell growth, survival, proliferation, motility and morphology. Involved in the activation of AKT1 upon stimulation by G-protein coupled receptors (GPCRs) ligands such as CXCL12, sphingosine 1-phosphate, and lysophosphatidic acid. May also act downstream receptor tyrosine kinases. Required in different signaling pathways for stable platelet adhesion and aggregation. Plays a role in platelet activation signaling triggered by GPCRs, alpha-IIb/beta-3 integrins (ITGA2B/ ITGB3) and ITAM (immunoreceptor tyrosine-based activation motif)-bearing receptors such as GP6. Regulates the strength of adhesion of ITGA2B/ ITGB3 activated receptors necessary for the cellular transmission of contractile forces. Required for platelet aggregation induced by F2 (thrombin) and thromboxane A2 (TXA2). Has a role in cell survival. May have a role in cell migration. Involved in the early stage of autophagosome formation. Modulates the intracellular level of PtdIns3P (phosphatidylinositol 3-phosphate) and activates PIK3C3 kinase activity. May act as a scaffold, independently of its lipid kinase activity to positively regulate autophagy. May have a role in insulin signaling as scaffolding protein in which the lipid kinase activity is not required. May have a kinase-independent function in regulating cell proliferation and in clathrin-mediated endocytosis. Mediator of oncogenic signal in cell lines lacking PTEN. The lipid kinase activity is necessary for its role in oncogenic transformation. Required for the growth of ERBB2 and RAS driven tumors. Has also a protein kinase activity showing autophosphorylation (PubMed:12502714).

GENE ID 5291
SWISS PROT P42338
SYNONYMS PI3K; PI3Kbeta; PIK3C1; p110-BETA; Phosphatidylinositol 4,5-bisphosphate 3-kinase 110 kDa catalytic subunit beta


Materials Supplied

Kit Components 96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate 1 plate
Plate sealer for 96 wells 2
Standard
2 tubes
Diluent buffer 1 bottle
Detection Reagent A 1 bottle
Detection Reagent B 1 bottle
TMB Substrate 1 tube
Stop Solution 1 tube
Wash Buffer (30 ℅ concentrate) 1 tube
Product data sheet 1 copy

Storage

Storage The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY

Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level PIK3Cb were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level PIK3Cb were tested on 3 different plates, with 8 replicates in each plate.
CV(%) = SD/meanX100

Intra-Assay: CV<10%
Inter-Assay: CV<12%

SENSITIVITY The minimum detectable dose was 0.059ng/mL.
ASSAY RANGE 0.156-10ng/mL
SPECIFICITY This assay has high sensitivity and excellent specificity for the detection of PIK3Cb.
No significant cross-reactivity or interference between PIK3Cb and analogs was observed.
Note:
Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between PIK3Cb and all analogs, therefore, cross reactivity may still exist.