Recombinant Human SPINK1 Protein, CF Summary
Product Specifications
Asp24-Cys79, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
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.
|
Assay Procedure
- 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
- Dilute Trypsin to 0.25 µg/mL in Assay Buffer.
- 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.
- 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.
- Incubate reaction mixtures at room temperature for 30 minutes.
- After incubation, dilute reaction mixtures 5-fold with Assay Buffer. (Example: 40 µL reaction mixture + 160 µL Assay Buffer).
- Dilute Substrate to 20 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 320 nM and 405 nM (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) value for rhSPINK1 by plotting final concentration per well of rhSPINK1 vs. specific activity with 4-PL fitting.
- 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
Reconstitution Calculator
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).
- Deybach J.C.D. et al. (2003) Hum. Genet. 113:369.
- Chandak G.R. et al. (2002) J. Med. Genet. 39:347.
- Huhtala M.L. et al. (1982) J. Biol. Chem. 257:13713.
Citations for Recombinant Human SPINK1 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Raised SPINK1 levels play a role in angiogenesis and the transendothelial migration of ALL cells
Authors: D Luo, D Liu, C Rao, S Shi, X Zeng, S Liu, H Jiang, L Liu, Z Zhang, X Lu
Scientific Reports, 2022-02-22;12(1):2999.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Role of trypsin and protease-activated receptor-2 in ovarian cancer
Authors: KK Kim, R Turner, N Khazan, A Kodza, A Jones, RK Singh, RG Moore
PLoS ONE, 2020-05-04;15(5):e0232253.
Species: N/A
Sample Types: Protein
Applications: Bioassay
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