Recombinant Human Lipocalin-1 Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human Lipocalin-1 Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit active Cathepsin V cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ES008). The IC₅₀ value is <400 nM, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Lipocalin-1 protein
  His19-Asp176, with a C-terminal 10-His tag
• Accession #: P31025
• N-terminal Sequence: His19
• Protein/Peptide Type: Recombinant Enzymes
• Gene: LCN1
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 19 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 20-22 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Sodium Acetate, 0.15 M NaCl, pH 5.5
  • Cathepsin Buffer: 50 mM Sodium Acetate, 0.15 M NaCl, 10 mM DTT, pH 5.5
  • Substrate Buffer: 50 mM Sodium Acetate, 0.15 M NaCl, 5 mM DTT, pH 5.5
  • Recombinant Human Lipocalin-1 (rhLipocalin-1) (Catalog # 1708-PI)
  • Recombinant Human Cathepsin V (rhCathepsin V) (Catalog # 1080-CY)
  • Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Prepare a curve of rhLipocalin-1 (MW: 18,805 Da) in Assay Buffer. Make the following serial dilutions: 40,000, 10,000, 4000, 1000, 500, 250, 125, 50, 12.5, and 6.25 nM.
  2. Dilute rhCathepsin V to 2 µg/mL in Cathepsin Buffer.
  3. Mix equal volumes of the rhLipocalin-1 curve dilutions and the 2 µg/mL rhCathepsin V. Include a Cathepsin Control (in duplicate) containing Assay Buffer in place of rhLipocalin-1.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. Dilute Substrate to 40 µM in Substrate Buffer.
  6. Load into a black well plate 50 µL of the incubated mixtures, and start the reaction by adding 50 µL of 40 µM Substrate.
  7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  8. Derive the 50 % inhibiting concentration (IC₅₀) of rhLipocalin-1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
  9. The specific activity for rhCathepsin V at each point may be determined 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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

  Per Well:
  • rhCathepsin V: 0.05 µg
  • rhLipocalin-1: 10,000, 2500, 1000, 250, 125, 62.5, 31.25, 12.5, 3.125, and 1.5625 nM
  • Substrate: 20 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Lipocalin-1 Protein, CF

• LCN1
• lipocalin 1 (tear prealbumin)
• lipocalin 1-like 2
• Lipocalin1
• Lipocalin-1
• MGC71975
• PMFA
• Tear lipocalin
• Tear Prealbumin
• TLC
• TPVon Ebner gland protein
• VEG protein
• VEGP
• VEGPlipocalin 1 (protein migrating faster than albumin, tear prealbumin)

Background

Lipocalin-1, also known as tear prealbumin or von Ebner’s gland protein (VEGP), is encoded by the LCN1 gene (1-3). It is a member of the Lipocalin superfamily that binds many different classes of lipophylic chemicals (4). Lipocalin-1 contains three sequence motifs similar to the cystatins, a superfamily of cysteine protease inhibitors (5). In fact, it has been suggested that Lipocalin-1 is a physiological inhibitor of cysteine proteases and plays a role in the control of inflammatory processes in oral and ocular tissues (5). Recombinant Human Lipocalin-1 corresponds to the mature and secreted protein. It is a weak inhibitor of cysteine proteases such as cathepsin V, which is similar to recombinant human Cystatin S (Catalog # 1296-PI).

1. Redl, B. et al. (1992) J. Biol. Chem. 267:20282.
2. Blaker, M. et al. (1993) Biochim. Biophys. Acta 1172:131.
3. Lassagne, H. and A.M. Gachon (1993) Exp. Eye Res. 56:605.
4. Redl, B. et al. (2000) Biochim. Biophys. Acta 1482:241.
5. van’t Hof, W. et al. (1997) J. Biol. Chem. 272:1837.

Publications for Lipocalin-1 (1708-PI)(2)

Publications using 1708-PI

• SK Chintala, J Pan, S Satapathy, R Condruti, Z Hao, PW Liu, CF O'Conner, JT Barr, MR Wilson, S Jeong, ME Fini Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy International Journal of Molecular Sciences, 2023-01-04;24(2):. 2023-01-04 [PMID: 36674497] (In Vivo, Mouse)
• Masoudi S, Zhong L, Raftery M, Stapleton F, Willcox M Method development for quantification of five tear proteins using selected reaction monitoring (SRM) mass spectrometry. Invest Ophthalmol Vis Sci, 2014-02-10;55(2):767-75. 2014-02-10 [PMID: 24408985] (Mass Spectrometry)

Bioinformatics

• Gene Symbol: LCN1
• Uniprot:
  • P31025()