Recombinant Human Napsin A Protein, CF
Recombinant Human Napsin A Protein, CF Summary
Thr25-Gly420, with C-terminal 6-His tag
|Formulation||Supplied as a 0.2 μm filtered solution in MES and NaCl.|
|Shipping||The product is shipped with polar packs. 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: 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
- Dilute rhNAPSA to 40 µg/mL in Assay Buffer.
- Incubate 50 µL aliquot of 40 µg/mL rhNAPSA at room temperature for 20 minutes.
- Dilute incubated rhNAPSA to 8 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
- 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
Background: Napsin A
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).
- Schauer-Vukasinovic, V. et al. (2001) Biochim. Biophys. Acta 1524:51.
- Tatnell, P.J. et al. (1998) FEBS Lett. 441:43.
- Schauer-Vukasinovic, V. et al. (1999) FEBS Lett. 462:135.
- Yamashita, Y. et al. (2014) Mod. Pathol. PMID 24721826
- Skirnisdottir, I. et al. (2013) BMC Cancer. 13:524.
- Ordonez, N.G. (2012) Adv. Anat. Pathol. 19:66.
- Jagirdar, J. (2008) Arch. Pathol. Lab. Med. 132:384.
- Ao, M.H. et al. (2014) Hum. Pathol. 45:926.
- Brasch, F. et al. (2003) J. Biol. Chem. 278:49006.
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