Recombinant Human Napsin A Protein, CF

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

Recombinant Human Napsin A Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH₂ (Catalog # ES001). The specific activity is >12 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Napsin A protein
  Thr25-Gly420, with C-terminal 6-His tag
• Accession #: O96009
• N-terminal Sequence: Thr25
• Protein/Peptide Type: Recombinant Enzymes
• Gene: NAPSA
• 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:
  • Enzyme Activity
• Theoretical MW: 44 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: 40-70 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES and NaCl.
• Purity: >80%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 0.1 M NaOAc, 0.2 M NaCl, pH 3.5
  • Recombinant Human Napsin A (rhNAPSA) (Catalog # 8489-NA)
  • Substrate: MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (Catalog # ES001), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhNAPSA to 40 µg/mL in Assay Buffer.
  2. Incubate 50 µL aliquot of 40 µg/mL rhNAPSA at room temperature for 20 minutes.
  3. Dilute incubated rhNAPSA to 8 µg/mL in Assay Buffer.
  4. Dilute Substrate to 20 µM in Assay Buffer.
  5. Load 50 µL incubated rhNAPSA into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 µL of 20 µM Substrate.
  6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

  Per Well:
  • rhNAPSA: 0.4 µg
  • Substrate: 10 µM

Notes

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

Alternate Names for Recombinant Human Napsin A Protein, CF

• Asp 4
• ASP4
• Aspartyl protease 4
• EC 3.4.23
• EC 3.4.23.-
• EC 3.4.23.15
• EC 3.4.23.3
• EC 3.4.23.5
• KAP
• Kdap
• NAP1napsin-A
• NAPA
• NAPApronapsin A
• NAPSA
• napsin A aspartic peptidase
• Napsin A
• Napsin-1
• SNAPA
• TA01/TA02

Background

Human NAPSA (also called Napsin A) is a 38 kDa glycosylated aspartic protease (1-3). NAPSA/Napsin A is synthesized as a 420 amino acid (aa) proenzyme containing a signal peptide (up to 25 aa), an activation peptide (38 aa), and a 357 aa mature enzyme. Mature human NAPSA/Napsin A shares 74% and 73% aa sequence identity with mature mouse and rat NAPSA/Napsin A, respectively. The NAPSA/Napsin A proenzyme is expressed in lung epithelial cells and within the kidney (3). Kidney dysfunction is associated with a decrease in NAPSA/Napsin A excretion in urine (1). NAPSA/Napsin A expression is also used as a marker for clear cell ovarian carcinomas, pulmonary adenocarcinomas, and renal cell carcinomas (4-8). NAPSA/Napsin A is involved in processing surfactant protein B in type II pneumocytes (9).

1. Schauer-Vukasinovic, V. et al. (2001) Biochim. Biophys. Acta 1524:51.
2. Tatnell, P.J. et al. (1998) FEBS Lett. 441:43.
3. Schauer-Vukasinovic, V. et al. (1999) FEBS Lett. 462:135.
4. Yamashita, Y. et al. (2014) Mod. Pathol. PMID 24721826
5. Skirnisdottir, I. et al. (2013) BMC Cancer. 13:524.
6. Ordonez, N.G. (2012) Adv. Anat. Pathol. 19:66.
7. Jagirdar, J. (2008) Arch. Pathol. Lab. Med. 132:384.
8. Ao, M.H. et al. (2014) Hum. Pathol. 45:926.
9. Brasch, F. et al. (2003) J. Biol. Chem. 278:49006.

Bioinformatics

• Gene Symbol: NAPSA
• Uniprot:
  • O96009()