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Human Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) ELISA Kit

SKU:
HUEB1519
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P42336
Range:
0.156-10 ng/mL
ELISA Type:
Sandwich
Synonyms:
PIK3CA, Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha isoform
Reactivity:
Human
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Description

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Product Name:Human Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) ELISA Kit
Product Code:HUEB1519
Alias:Phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha isoform, PI3-kinase subunit alpha, PI3K-alpha, PI3Kalpha, PtdIns-3-kinase subunit alpha, Phosphatidylinositol-4, 5-bisphosphate 3-kinase 110 kDa catalytic subunit alpha, PtdIns-3-kinase subunit p110-alpha, p110alpha, Phosphoinositide-3-kinase catalytic alpha polypeptide, Serine/threonine protein kinase PIK3CA, PIK3CA, 2.7.1.153, 2.7.11.1
Uniprot:P42336
Reactivity:Human
Range:0.156-10 ng/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:PIK3CA: Phosphoinositide-3-kinase (PI3K) that phosphorylates PtdIns (Phosphatidylinositol), PtdIns4P (Phosphatidylinositol 4- phosphate) and PtdIns(4,5)P2 (Phosphatidylinositol 4,5- bisphosphate) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). 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. Participates in cellular signaling in response to various growth factors. Involved in the activation of AKT1 upon stimulation by receptor tyrosine kinases ligands such as EGF, insulin, IGF1, VEGFA and PDGF. Involved in signaling via insulin-receptor substrate (IRS) proteins. Essential in endothelial cell migration during vascular development through VEGFA signaling, possibly by regulating RhoA activity. Required for lymphatic vasculature development, possibly by binding to RAS and by activation by EGF and FGF2, but not by PDGF. Regulates invadopodia formation in breast cancer cells through the PDPK1- AKT1 pathway. Participates in cardiomyogenesis in embryonic stem cells through a AKT1 pathway. Participates in vasculogenesis in embryonic stem cells through PDK1 and protein kinase C pathway. Has also serine-protein kinase activity: phosphorylates PIK3R1 (p85alpha regulatory subunit), EIF4EBP1 and HRAS. Heterodimer of a catalytic subunit PIK3CA and a p85 regulatory subunit (PIK3R1, PIK3R2 or PIK3R3). Interacts with IRS1 in nuclear extracts. Interacts with RUFY3. Interacts with RASD2. Interacts with APPL1. Interacts with HRAS1 and KRAS. Interaction with HRAS1/KRAS is required for PI3K pathway signaling and cell proliferation stimulated by EGF and FGF2. Belongs to the PI3/PI4-kinase family.
UniProt Protein Details:

Protein type:Motility/polarity/chemotaxis; Oncoprotein; EC 2.7.11.1; EC 2.7.1.153; Kinase, lipid; Carbohydrate Metabolism - inositol phosphate

Chromosomal Location of Human Ortholog: 3q26.3

Cellular Component: lamellipodium; plasma membrane; phosphoinositide 3-kinase complex; cytosol

Molecular Function:protein serine/threonine kinase activity; protein binding; phosphatidylinositol-4,5-bisphosphate 3-kinase activity; insulin receptor substrate binding; 1-phosphatidylinositol-3-kinase activity; kinase activity; protein kinase activator activity; ATP binding; phosphatidylinositol-4-phosphate 3-kinase activity; phosphoinositide 3-kinase activity

Biological Process: epidermal growth factor receptor signaling pathway; platelet activation; phosphoinositide-mediated signaling; fibroblast growth factor receptor signaling pathway; nerve growth factor receptor signaling pathway; regulation of multicellular organism growth; glucose metabolic process; positive regulation of peptidyl-serine phosphorylation; T cell receptor signaling pathway; protein amino acid phosphorylation; vasculature development; phosphoinositide phosphorylation; positive regulation of protein kinase activity; phospholipid metabolic process; T cell costimulation; phosphatidylinositol biosynthetic process; protein kinase B signaling cascade; insulin receptor signaling pathway; innate immune response; endothelial cell migration; negative regulation of neuron apoptosis; angiogenesis; blood coagulation; vascular endothelial growth factor receptor signaling pathway; leukocyte migration; cardiac muscle contraction

Disease: Cowden Syndrome 5; Gastric Cancer; Keratosis, Seborrheic; Breast Cancer; Lung Cancer; Ovarian Cancer; Congenital Lipomatous Overgrowth, Vascular Malformations, And Epidermal Nevi; Colorectal Cancer; Megalencephaly-capillary Malformation-polymicrogyria Syndrome; Hepatocellular Carcinoma; Nevus, Epidermal

NCBI Summary:Phosphatidylinositol 3-kinase is composed of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. The protein encoded by this gene represents the catalytic subunit, which uses ATP to phosphorylate PtdIns, PtdIns4P and PtdIns(4,5)P2. This gene has been found to be oncogenic and has been implicated in cervical cancers. A pseudogene of this gene has been defined on chromosome 22. [provided by RefSeq, Apr 2016]
UniProt Code:P42336
NCBI GenInfo Identifier:126302584
NCBI Gene ID:5290
NCBI Accession:P42336.2
UniProt Related Accession:P42336
Molecular Weight:~ 110kDa
NCBI Full Name:Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
NCBI Synonym Full Names:phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha
NCBI Official Symbol:PIK3CA  
NCBI Official Synonym Symbols:MCM; CWS5; MCAP; PI3K; CLAPO; CLOVE; MCMTC; PI3K-alpha; p110-alpha  
NCBI Protein Information:phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
UniProt Protein Name:Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform
UniProt Synonym Protein Names:Phosphatidylinositol 4,5-bisphosphate 3-kinase 110 kDa catalytic subunit alpha; PtdIns-3-kinase subunit p110-alpha; p110alpha; Phosphoinositide-3-kinase catalytic alpha polypeptide; Serine/threonine protein kinase PIK3CA (EC:2.7.11.1)
UniProt Gene Name:PIK3CA  
UniProt Entry Name:PK3CA_HUMAN
Component Quantity (96 Assays) Storage
ELISA Microplate (Dismountable) 8×12 strips -20°C
Lyophilized Standard 2 -20°C
Sample Diluent 20ml -20°C
Assay Diluent A 10mL -20°C
Assay Diluent B 10mL -20°C
Detection Reagent A 120µL -20°C
Detection Reagent B 120µL -20°C
Wash Buffer 30mL 4°C
Substrate 10mL 4°C
Stop Solution 10mL 4°C
Plate Sealer 5 -

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1. Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2. Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5. Repeat the wash process for five times as conducted in step 3.
6. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7. Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9. After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol
Serum If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
ELISA
Human PIK3CA ELISA Kit

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