Recombinant Human SPINK1 Protein, CF

R&D Systems | Catalog # 7496-PI

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human SPINK1 Protein (7496-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P00995

Applications

Enzyme Activity
View all SPINK1 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human SPINK1 protein
Asp24-Cys79, with an N-terminal Met and 6-His tag

Purity

>85%, 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.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

7 kDa

SDS-PAGE

8-11 kDa, reducing conditions

Activity

Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002).
The IC50 value is <10 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

7496-PI
Formulation Supplied as a 0.2 μm filtered solution in MES, NaCl, CaCl2, DTT, Glycerol and Brij-35.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: SPINK1

SPINK1 is a serine protease inhibitor with one Kazal-like domain that is secreted  by acinar cells of the pancreas.  It is an inhibitor of trypsin that prevents the premature activation of protease zymogens in the pancreas.  Deficiencies in SPINK1 are a cause of hereditary pancreatitis and tropical calcific pancreatitis (1, 2).  Because elevated SPINK1 expression has been observed with several tumor types, the protein is also known as the tumor-associated trypsin inhibitor (3).

References

  1. Deybach J.C.D. et al. (2003) Hum. Genet. 113:369.
  2. Chandak G.R. et al. (2002) J. Med. Genet. 39:347.
  3. Huhtala M.L. et al. (1982) J. Biol. Chem. 257:13713.

Long Name

Serine Peptidase Inhibitor, Kazal Type 1

Alternate Names

PCTT, PSTI, Spink3, TATI

Entrez Gene IDs

6690 (Human); 20730 (Mouse); 266602 (Rat)

Gene Symbol

SPINK1

UniProt

P00995

Additional SPINK1 Products

Product Documents for Recombinant Human SPINK1 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human SPINK1 Protein, CF

For research use only

Citations for Recombinant Human SPINK1 Protein, CF

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Protocols

View specific protocols for Recombinant Human SPINK1 Protein, CF (7496-PI):

Materials
  • Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
  • Recombinant Human SPINK1 (rhSPINK1) (Catalog # 7496-PI)
  • Trypsin (Sigma, Catalog # T-1426)
  • Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute Trypsin to 0.25 µg/mL in Assay Buffer.
  2. Prepare a curve of rhSPINK1 (MW: 7201 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 nM.
  3. Combine equal volumes of 0.25 µg/mL Trypsin with rhSPINK1 serial curve dilutions. Include two controls containing 0.25 µg/mL Trypsin with Assay Buffer.
  4. Incubate reaction mixtures at room temperature for 30 minutes.
  5. After incubation, dilute reaction mixtures 5-fold with Assay Buffer. (Example: 40 µL reaction mixture + 160 µL Assay Buffer).
  6. Dilute Substrate to 20 µM in Assay Buffer.
  7. In a plate, load 50 µL of the diluted reaction mixtures, and start the reaction by adding 50 µL of 20 µM Substrate to wells.
  8. Read at excitation and emission wavelengths of 320 nM and 405 nM  (top read), respectively, in kinetic mode for 5 minutes.
  9. Derive the 50% inhibiting concentration (IC50) value for rhSPINK1 by plotting final concentration per well of rhSPINK1 vs. specific activity with 4-PL fitting.
  10. Calculate specific activity for each point using the following formula (if needed):

     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-P-L-OH (Bachem, Catalog # M-1975).

  • Trypsin: 0.00125 µg
  • rhSPINK1 curve: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391 and 0.195 nM
  • Substrate: 10 µM

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