Recombinant Human PTEN Protein, CF Summary
Thr2-Val403, with a C-terminal 6-His tag
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CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in HEPES, NaCl, EDTA, Glycerol and Betamercaptoethanol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 100 mM Tris, 2 mM DTT, 0.05% (v/v) NP-40 Substitute (Sigma, Catalog # 74385), pH 8.5
- Vesicle Buffer: 10 mM HEPES, 1 mM EGTA and 1 mg/mL BSA, pH 7.5
- Recombinant Human PTEN (rhPTEN) (Catalog # 847-PN)
- Substrate: diC16PIP3 (Echelon, Catalog # P-3916)
- Lipids: dioleoyl phosphatidylcholine (DOPC), and dioleoyl phosphatidylserine (DOPS) (Avanti Polar Lipids, Catalog # 790595)
- Malachite Green Phosphate Detection Kit (R&D Systems, Catalog # DY996)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare a solution of 12.5 mg/mL DOPC, 12.5 mg/mL DOPS in Vesicle Buffer.
- Form Phospholipid Vesicles by combining substrate and lipids in Vesicle Buffer to final concentrations of 0.3 mM diC16PIP3, 625 μg/mL DOPC, 625 μg/mL DOPS. Mix vigorously.
- Dilute rhPTEN to 0.25 ng/μL in Assay Buffer.
- Dilute Phospholipid Vesicles to 0.09 mM diC16PIP3, 187.5 μg/mL DOPC, 187.5 μg/mL DOPS in Assay Buffer.
- Prepare a standard curve from the 1 M Phosphate Standard supplied in the malachite green phosphate detection kit. First, add 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock. Then, add 10 µL of the 10 mM phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. (This is the first dilution to use as a standard.) Finally, perform six additional two-fold serial dilutions of the 100 µM phosphate stock.
- Load 100 μL of each point of the curve to wells in triplicate.
- To the experimental wells, add 80 µL of 0.25 ng/μL rhPTEN. To the control wells, add 80 µL of Assay Buffer.
- Start the reaction by adding 20 µL of dilute Phospholipid Vesicles.
- Incubate at 37 ºC for 15 minutes.
- Add 20 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 20 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (nmol/min/mg) =
|Phosphate released* (nmol)|
|Incubation time (min) x amount of enzyme (mg)|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control wells.Per Reaction:
- rhPTEN: 20 ng = 0.00002 mg
- Substrate: 18 µM
- Standard Curve: 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10.0 nmol
The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10), also known as MMAC1 (mutated in multiple advanced cancers 1), encodes a phosphatase that contains the catalytic signature motif (HCXXGXXRS/T) found in all members of the protein tyrosine phosphatase family. In vitro, the recombinant PTEN has both lipid phosphatase and protein phosphatase activities (1, 2). Interestingly, accumulating evidence has shown that the tumor suppressor activity of PTEN relies on its ability to dephosphorylate phosphatidylinositol (3, 4, 5)-triphosphate specifically at position 3 of the inositol ring (3). This activity reduces the levels of phosphatidylinositol (3, 4, 5)-triphosphate which is specifically produced from phosphatidylinositol (4, 5)-diphosphate by PI 3-kinase upon activation by a variety of stimuli. Therefore, PTEN antagonizes PI 3-kinase-induced downstream signaling events and cellular processes including cell growth, apoptosis and cell motility. In vivo, the importance of PTEN catalytic activity in its tumor suppressor functions is underscored by the fact that the majority of PTEN missense mutations detected in tumor specimens target the phosphatase domain and cause a loss in PTEN phosphatase activity (4).
- Maehama, T. and J. Dixon (1998) J. Biol. Chem. 273:13375.
- Das, S. et al. (2003) Proc. Natl. Acad. Sci. USA 100:7491.
- Myers, M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:13513.
- Waite, K. and C. Eng (2002) Am. J. Hum. Genet. 70:829.
Product Specific NoticesThis product is covered by one or more of the following U.S. patents: 6,262,242, 6,482,795, and U.S. patent application serial number 10/299,003.
Citations for Recombinant Human PTEN Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 3
Filter your results:
PTEN regulates invasiveness in pancreatic neuroendocrine tumors through DUSP19-mediated VEGFR3 dephosphorylation
Authors: TM Chang, PY Chu, HY Lin, KW Huang, WC Hung, YS Shan, LT Chen, HJ Tsai
Journal of Biomedical Science, 2022;29(1):92.
Sample Types: Peptide
The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein
Authors: M Chen, DG Nowak, N Narula, B Robinson, K Watrud, A Ambrico, TM Herzka, ME Zeeman, M Minderer, W Zheng, SH Ebbesen, KS Plafker, C Stahlhut, VM Wang, L Wills, A Nasar, M Castillo-M, C Cordon-Car, JE Wilkinson, S Powers, R Sordella, NK Altorki, V Mittal, BM Stiles, SM Plafker, LC Trotman
J. Cell Biol, 2017;0(0):.
Protein kinase Czeta-dependent LKB1 serine 428 phosphorylation increases LKB1 nucleus export and apoptosis in endothelial cells.
Authors: Song P, Xie Z, Wu Y, Xu J, Dong Y, Zou MH
J. Biol. Chem., 2008;283(18):12446-55.
Sample Types: N/A
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Phosphatase Activity Assays
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