Recombinant Human PTEN Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

Order Details

Recombinant Human PTEN Protein, CF Summary

Details of Functionality
Measured by its ability to dephosphorylate either diC8PIP3 or diC16PIP3. The specific activity is >480 nmol/min/mg, as measured under the described conditions.
Source
E. coli-derived human PTEN protein
Thr2-Val403, with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Thr2
Protein/Peptide Type
Recombinant Enzymes
Gene
PTEN
Purity
>80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Dilutions
  • Inhibition Activity
Theoretical MW
48 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
59 kDa, reducing conditions
Publications
Read Publications using
847-PN in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in HEPES, NaCl, EDTA, Glycerol and Betamercaptoethanol.
Purity
>80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
  • 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
  1. Prepare a solution of 12.5 mg/mL DOPC, 12.5 mg/mL DOPS in Vesicle Buffer.
  2. 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.
  3. Dilute rhPTEN to 0.25 ng/μL in Assay Buffer.
  4. Dilute Phospholipid Vesicles to 0.09 mM diC16PIP3, 187.5 μg/mL DOPC, 187.5 μg/mL DOPS in Assay Buffer.
  5. 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.
  6. Load 100 μL of each point of the curve to wells in triplicate.
  7. To the experimental wells, add 80 µL of 0.25 ng/μL rhPTEN. To the control wells, add 80 µL of Assay Buffer.
  8. Start the reaction by adding 20 µL of dilute Phospholipid Vesicles.
  9. Incubate at 37 ºC for 15 minutes.
  10. Add 20 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  11. Add 20 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. 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

Notes

This 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.



This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human PTEN Protein, CF

  • BZS
  • EC 3.1.3.16,10q23del
  • EC 3.1.3.48
  • EC 3.1.3.67
  • GLM2
  • MMAC1 phosphatase and tensin homolog deleted on chromosome 10
  • MMAC1
  • MMAC1MGC11227
  • Mutated in multiple advanced cancers 1
  • phosphatase and tensin homologDEC
  • phosphatidylinositol-34,5-trisphosphate 3-phosphatase and dual-specificityprotein phosphatase PTEN
  • PTEN
  • PTEN1
  • TEP1MHAM

Background

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).

  1. Maehama, T. and J. Dixon (1998) J. Biol. Chem. 273:13375.
  2. Das, S. et al. (2003) Proc. Natl. Acad. Sci. USA 100:7491.
  3. Myers, M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:13513.
  4. Waite, K. and C. Eng (2002) Am. J. Hum. Genet. 70:829.

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Publications for PTEN (847-PN)(2)

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Bioinformatics

Gene Symbol PTEN
Uniprot