Human Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN (PTEN) ELISA Kit
- SKU:
- HUEB1595
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P60484
- Range:
- 0.156-10 ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- PTEN, Phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN, Mutated in multiple advanced cancers 1, Phosphatase and tensin homolog, MMAC1, TEP1
- Reactivity:
- Human
Description
| Product Name: | Human Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN (PTEN) ELISA Kit |
| Product Code: | HUEB1595 |
| Alias: | Phosphatidylinositol-3, 4, 5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN, Mutated in multiple advanced cancers 1, Phosphatase and tensin homolog, PTEN, MMAC1, TEP1, 3.1.3.16 |
| Uniprot: | P60484 |
| 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: | PTEN: a phosphoinositide 3-phosphatase (PIP3) and a tumor suppressor implicated in a wide variety of human cancers. Dephosphorylates inositol phospholipids generated by the activation of the phosphoinositide 3 kinase (PI3K). A major negative regulator of the PI3K/Akt signaling pathway. Possesses a C-terminal regulatory domain that contains three phosphorylation sites which may regulate its stability. |
| UniProt Protein Details: | Protein type:Carbohydrate Metabolism - inositol phosphate; EC 3.1.3.48; Phosphatase, lipid; Nuclear receptor co-regulator; Motility/polarity/chemotaxis; EC 3.1.3.16; EC 3.1.3.67; Tumor suppressor Chromosomal Location of Human Ortholog: 10q23.3 Cellular Component: nucleoplasm; postsynaptic membrane; PML body; internal side of plasma membrane; neuron projection; cell projection; mitochondrion; cytoplasm; plasma membrane; extracellular region; dendritic spine; cytosol; nucleus Molecular Function:phosphatidylinositol-3-phosphatase activity; phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase activity; inositol-1,3,4,5-tetrakisphosphate 3-phosphatase activity; platelet-derived growth factor receptor binding; magnesium ion binding; protein tyrosine phosphatase activity; protein serine/threonine phosphatase activity; protein kinase binding; PDZ domain binding; protein binding; enzyme binding; phosphatidylinositol-3,4-bisphosphate 3-phosphatase activity; protein tyrosine/serine/threonine phosphatase activity; phosphoprotein phosphatase activity; lipid binding Biological Process: central nervous system development; male mating behavior; nerve growth factor receptor signaling pathway; prepulse inhibition; response to arsenic; regulation of cellular component size; heart development; anaphase-promoting complex activation during mitotic cell cycle; Wnt receptor signaling pathway through beta-catenin; T cell receptor signaling pathway; regulation of B cell apoptosis; myelin maintenance in the central nervous system; response to glucose stimulus; negative regulation of axonogenesis; response to drug; rhythmic synaptic transmission; fibroblast growth factor receptor signaling pathway; dentate gyrus development; negative regulation of myelination; social behavior; forebrain morphogenesis; negative regulation of organ growth; response to ethanol; response to zinc ion; inositol phosphate dephosphorylation; phosphatidylinositol biosynthetic process; endothelial cell migration; positive regulation of transcription factor activity; multicellular organismal response to stress; negative regulation of apoptosis; negative regulation of phagocytosis; synapse maturation; inositol phosphate metabolic process; apoptosis; regulation of protein stability; locomotory behavior; negative regulation of cell size; platelet-derived growth factor receptor signaling pathway; protein amino acid dephosphorylation; regulation of myeloid cell apoptosis; response to estradiol stimulus; negative regulation of cell proliferation; locomotor rhythm; learning and/or memory; synaptogenesis; negative regulation of protein amino acid phosphorylation; phospholipid metabolic process; positive regulation of cell proliferation; brain morphogenesis; protein kinase B signaling cascade; nerve-nerve synaptic transmission; angiogenesis; negative regulation of cell migration; response to nutrient; negative regulation of epithelial cell proliferation; aging; cardiac muscle development; epidermal growth factor receptor signaling pathway; cell migration; phosphoinositide-mediated signaling; negative regulation of protein kinase B signaling cascade; protein stabilization; central nervous system neuron axonogenesis; maternal behavior; negative regulation of focal adhesion formation; response to ATP; memory; cell proliferation; G1/S-specific negative regulation of cyclin-dependent protein kinase activity; innate immune response; phosphoinositide dephosphorylation; regulation of cyclin-dependent protein kinase activity; negative regulation of phosphoinositide 3-kinase cascade Disease: Meningioma, Familial, Susceptibility To; Thyroid Carcinoma, Follicular; Melanoma, Cutaneous Malignant, Susceptibility To, 1; Bannayan-riley-ruvalcaba Syndrome; Cowden Syndrome 1; Squamous Cell Carcinoma, Head And Neck; Prostate Cancer; Glioma Susceptibility 2; Vacterl Association With Hydrocephalus; Endometrial Cancer; Macrocephaly/autism Syndrome |
| NCBI Summary: | This gene was identified as a tumor suppressor that is mutated in a large number of cancers at high frequency. The protein encoded this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase. It contains a tensin like domain as well as a catalytic domain similar to that of the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate in cells and functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P60484 |
| NCBI GenInfo Identifier: | 42560209 |
| NCBI Gene ID: | 5728 |
| NCBI Accession: | P60484.1 |
| UniProt Secondary Accession: | P60484,O00633, O02679, Q6ICT7, B2R904, F2YHV0, |
| UniProt Related Accession: | P60484 |
| Molecular Weight: | 19,796 Da |
| NCBI Full Name: | Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN |
| NCBI Synonym Full Names: | phosphatase and tensin homolog |
| NCBI Official Symbol: | PTEN |
| NCBI Official Synonym Symbols: | BZS; DEC; CWS1; GLM2; MHAM; TEP1; MMAC1; PTEN1; 10q23del |
| NCBI Protein Information: | phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN; mitochondrial PTENalpha; phosphatase and tensin-like protein; mutated in multiple advanced cancers 1; mitochondrial phosphatase and tensin protein alpha; MMAC1 phosphatase and tensin homolog deleted on chromosome 10; phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN |
| UniProt Protein Name: | Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN |
| UniProt Synonym Protein Names: | Mutated in multiple advanced cancers 1; Phosphatase and tensin homolog |
| Protein Family: | Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase |
| UniProt Gene Name: | PTEN |
| UniProt Entry Name: | PTEN_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. |
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