Recombinant Human GSTP1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human GSTP1 Protein, CF Summary

• Details of Functionality: Measured by the conjugation of reduced glutathione to 1-bromo-2,4-dinitrobenzene. The specific activity is >25,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Glutathione S-Transferase pi 1/GSTP1 protein
  Met1-Glu210
• Accession #: AAC51280
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GSTP1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 23 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: 24 kDa, reducing conditions

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 Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 100 mM NaH₂PO₄, pH 7.0
  • Recombinant Human Glutathione S‑Transferase pi 1/GSTP1 (rhGSTP1) (Catalog # 6455-GT)
  • Substrate: 1-bromo-2,4-dinitrobenzene (BDNB) (Sigma, Catalog # 262226), 75 mM stock in ethanol
  • L-Glutathione, reduced (GSH) (Amresco, Catalog # 399), 250 mM stock in deionized water
  • UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhGSTP1 to 0.2 ng/μL in Assay Buffer.
  2. Dilute GSH to 4 mM in Assay Buffer.
  3. Combine equal volumes of 0.2 ng/μL rhGSTP1 and 4 mM GSH for 0.1 ng/μL rhGSTP1 with 2 mM GSH.
  4. Dilute Substrate to 2 mM in Assay Buffer.
  5. Load into a UV plate 50 μL of the rhGSTP1/GSH mixture. Include a substrate blank containing 25 μL of Assay Buffer with 25 μL of the 4 mM GSH prepared in step 2.
  6. Start the reaction by adding 50 μL of 2 mM Substrate to well.
  7. Read in kinetic mode for 5 minutes at an absorbance of 340 nm.
  8. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 9600 M^-1cm^-1
  ***Using the path correction 0.320 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rhGSTP1: 0.005 μg
  • GSH: 1 mM
  • Substrate: 1 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human GSTP1 Protein, CF

• deafness, X-linked 7
• DFN7
• EC 2.5.1.18
• FAEES3
• fatty acid ethyl ester synthase III
• glutathione S-transferase P
• Glutathione STransferase pi 1
• Glutathione S-Transferase pi 1
• GST class-pi
• GST3DFN7
• GSTP
• GSTP1
• GSTP1-1
• PI

Background

Glutathione S-Transferases (GSTs) are members of the phase II detoxification enzyme family that conjugate glutathione to various electrophilic compounds, including metabolites generated by oxidative processes in the body, environmental toxins or carcinogens, and anti-cancer drugs. GSTP1 is a cytosolic protein that belongs to pi class of the GST superfamily. It is crystallized as a homodimer (1), but also exists in solution as an equilibrium mixture of monomer and dimer, depending on the protein concentration (2). Four genetic variants of GSTP1 with different enzymatic activities have been identified, which indicates the particular allelic form expressed in tissues could contribute to variation in catalytic efficiency and biological functions (3, 4). Human GSTP1 is present at elevated levels in many tumor cells, and has unique properties as a cancer marker (5). Genetic polymorphisms and expression patterns of GSTP1 have been associated with a variety of effects on human cancer, anti-cancer drug resistance, and asthma (6). In addition to its role as a drug-metabolizing enzyme, GSTP1 has ligand binding properties and regulates kinase signaling pathways through protein-protein interactions (7).

1. Reinemer, P. et al. (1992) J. Mol. Biol. 227:214.
2. Huang, Y.C. et al. (2008) J. Biol. Chem. 283:32880.
3. Ali-Osman, F. et al. (1997) J. Biol. Chem. 272:10004.
4. Hu, X. et al. (1998) Cancer Res. 58:5340.
5. Sato, K. et al. (1992) Tohoku J. Exp. Med. 168:97.
6. Townsend, D.M. and K.D. Tew (2003) Oncogene 22:7369.
7. Adler, V. et al. (1999) EMBO J. 18:1321.

Bioinformatics

• Gene Symbol: GSTP1
• Uniprot:
  • AAC51280()