Recombinant Human Napsin A Protein, CF Summary
Thr25-Gly420, with C-terminal 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|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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Fluorogenic Peptide Substrates
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