Recombinant Human Lipocalin-1 Protein, CF Summary
His19-Asp176, with a C-terminal 10-His tag
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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 Tris 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: 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
- 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.
- Dilute rhCathepsin V to 2 µg/mL in Cathepsin Buffer.
- 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.
- Incubate mixtures at room temperature for 30 minutes.
- Dilute Substrate to 40 µM in Substrate Buffer.
- Load into a black well plate 50 µL of the incubated mixtures, and start the reaction by adding 50 µL of 40 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50 % inhibiting concentration (IC50) of rhLipocalin-1 by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
- 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).
- 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
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).
- Redl, B. et al. (1992) J. Biol. Chem. 267:20282.
- Blaker, M. et al. (1993) Biochim. Biophys. Acta 1172:131.
- Lassagne, H. and A.M. Gachon (1993) Exp. Eye Res. 56:605.
- Redl, B. et al. (2000) Biochim. Biophys. Acta 1482:241.
- van’t Hof, W. et al. (1997) J. Biol. Chem. 272:1837.
Citation for Recombinant Human Lipocalin-1 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.
1 Citation: Showing 1 - 1
Method development for quantification of five tear proteins using selected reaction monitoring (SRM) mass spectrometry.
Authors: Masoudi S, Zhong L, Raftery M, Stapleton F, Willcox M
Invest Ophthalmol Vis Sci, 2014;55(2):767-75.
Sample Types: N/A
Applications: Mass Spectrometry
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Fluorogenic Peptide Substrates
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