Recombinant Human Napsin A Protein, CF

  
  • Purity
    >80%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
  • Endotoxin Level
    <1.0 EU per 1 μg of the protein by the LAL method.
  • Activity
    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. See Activity Assay Protocol on www.RnDSystems.com.
  • Source
    Chinese Hamster Ovary cell line, CHO-derived Thr25-Gly420, with C-terminal 6-His tag
  • Accession #
  • N-terminal Sequence
    Analysis
    Thr25
  • Predicted Molecular Mass
    44 kDa
  • SDS-PAGE
    40-70 kDa
8489-NA
 
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Assay Procedure
Materials
  • 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
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).
  • References:
    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.
  • Entrez Gene IDs:
    9476 (Human); 16541 (Mouse); 60575 (Rat)
  • Alternate Names:
    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
Related Research Areas
Fluorogenic Peptide Substrates
Description Application Cat# Citations Images  

Mca-PLGL-Dpa-AR-NH2 Fluorogenic MMP Substrate

BA, BA2, Bind ES001 13
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Primary Antibodies
Description Application Cat# Citations Images  

His Tag Antibody

WB, Simple Western, Flow, AP, CyTOF-ready MAB050 26  
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His Tag HRP-conjugated Antibody

WB, Simple Western MAB050H 3  
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His Tag APC-conjugated Antibody

ICFlow IC050A  
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His Tag Biotinylated Antibody

WB BAM050 4  
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His Tag PE-conjugated Antibody

ICFlow IC050P 2  
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His Tag Antibody

WB, Flow, AP MAB050R  
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His Tag Alexa Fluor® 488-conjugated Antibody

ICFlow IC050G  
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His Tag PerCP-conjugated Antibody

ICFlow IC050C  
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His Tag Alexa Fluor® 700-conjugated Antibody

ICFlow IC050N  
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Protein Purification Kits
Description Application Cat# Citations Images  

Histidine-tagged Protein Purification Resin

IP999 1
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