Recombinant Human UCH-L1/PGP9.5 Protein, CF

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

Recombinant Human UCH-L1/PGP9.5 Protein, CF Summary

• Details of Functionality: Measured by the hydrolysis of Ubiquitin-AMC. The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human UCH-L1/PGP9.5 protein
  Gln2-Ala223 with an N-terminal Met and 6-His tag
• Accession #: P09936
• N-terminal Sequence: Met
• Structure / Form: Monomer
• Protein/Peptide Type: Recombinant Enzymes
• Gene: UCHL1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 26 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: 27 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, Glycerol and DTT.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 0.5 mM EDTA, 1 mM DTT, 0.1 mg/mL Ovalbumin, pH 8.0
  • Recombinant Human UCH-L1/PGP9.5 (rhUCH-L1) (Catalog # 6007-CY)
  • Substrate: Ubiquitin-AMC, (Boston Biochem, Catalog # U-550), 50 µM in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhUCH-L1 to 0.5 µg/mL in Assay Buffer.
  2. Dilute Substrate to 2 µM in Assay Buffer.
  3. Load into a plate 50 µL of 0.5 µg/mL rhUCH-L1. Separately, also load 50 µL Assay Buffer to be used as a Substrate Blank.
  4. Seal plate and incubate both it and the diluted Substrate at 37 °C for 10 minutes. Also warm the plate reader to 37 °C.
  5. Start the reaction by adding 50 µL of 2 µM Substrate to all wells.
  6. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes.
  7. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (Sigma, Catalog # A9891).

  Per Well:
  • rhUCH-L1: 0.025 µg
  • Substrate: 1 µM

Notes

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

Alternate Names for Recombinant Human UCH-L1/PGP9.5 Protein, CF

• EC 3.4.19.12
• EC 6.-
• Neuron cytoplasmic protein 9.5
• PARK5
• PGP 9.5
• PGP9.5
• PGP9.5Uch-L1
• PGP95
• ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase)
• ubiquitin carboxyl-terminal hydrolase isozyme L1
• ubiquitin C-terminal hydrolase
• Ubiquitin thioesterase L1
• UCHL1
• UCH-L1

Background

Deubiquitination is a critical regulatory process in the ubiquitin-proteasome pathway (1). Ubiquitin C-terminal hydrolases (UCHs) are a family of cysteine proteases that catalyze the hydrolysis of a peptide bond at the C-terminal glycine of ubiquitin. Members of the UCH family have been implicated in a number of human diseases, including neurodegenerative diseases and cancers (2). Mutations of the UCH-L1 gene and alterations of the protein activity have been found to be associated with several neurodegenerative disorders, including Parkinson’s, Huntington’s and Alzheimer’s diseases (3).  It is also implicated in cancer tumorigenesis, including lung, breast, liver, kidney, colorectal and ovarian cancers (4-8). UCH-L1 is thought to be a tumor suppressor and biomarker for hepatocellular carcinoma and other digestive tumors.

1. Wing, S. (2003) Int. J. Biochem. Cell Biol. 35:590.
2. Ventii, KH and Wilkinson, KD (2008) Biochem. J. 414:161.
3. Gong, B. and Leznik, E. (2007) Drug News Perspect. 20:365.
4. Kim, H. et al. (2009) Oncogene 28:117.
5. Wang, W. et al. (2008) Int. J. Oncol. 33:1037.
6. Yu, J. et al. (2008) Hepatology 48:508.
7. Kagara, I. et al. (2008) J. Urol. 180:343.
8. Okochi-Takada, E. et al. (2006) Int. J. Cancer 119:1338.

Publications for UCH-L1/PGP9.5 (6007-CY)(2)

Publications using 6007-CY

• E Górska, M Tylicka, A Hermanowic, E Matuszczak, A Sankiewicz, E Gorodkiewi, J Hermanowic, E Karpi?ska, K Socha, J Kochanowic, M Jakoniuk, J Kami?ska, E Homšak, OM Koper-Lenk UCHL1, besides leptin and fibronectin, also could be a sensitive marker of the relapsing-remitting type of multiple sclerosis Scientific Reports, 2023-02-28;13(1):3423. 2023-02-28 [PMID: 36854961] (SPR, Human)
• S Bogdan, A Pu?cion-Ja, K Klimiuk, K Socha, J Kochanowic, E Gorodkiewi UCHL1 and Proteasome in Blood Serum in Relation to Dietary Habits, Concentration of Selected Antioxidant Minerals and Total Antioxidant Status among Patients with Alzheimer&#039;s Disease Journal of Clinical Medicine, 2022-01-14;11(2):. 2022-01-14 [PMID: 35054106] (Surface Plasmon Resonance (SPR, Human)

Bioinformatics

• Gene Symbol: UCHL1
• Uniprot:
  • P09936(Human)