Recombinant Mouse Cystatin F Protein, CF

Catalog #: 4557-PI Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
4557-PI-010

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Mouse Cystatin F Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to inhibit active Cathepsin L cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ES008). The IC₅₀ value is <60 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Cystatin F protein
Ala19-Gln144, with a C-terminal 6-His tag
Accession # O89098

Accession #

O89098

N-terminal Sequence
Analysis

Ala19

Structure / Form

Disulfide-linked homodimer

Predicted Molecular Mass

15 kDa

SDS-PAGE

20 kDa, reducing conditions

Product Datasheets

4557-PI

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

4557-PI

Formulation	Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
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: 50 mM MES, pH 6.0
Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock
Recombinant Mouse Cystatin F (rmCystatin F) (Catalog # 4557-PI)
Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
Substrate: Z-Leu-Arg-AMC (Catalog # ES008), 2 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rhCathepsin L to 40 µg/mL in Assay Buffer with 5 mM DTT.
Incubate on ice for 15 minutes.
After incubation, dilute activated rhCathepsin L to 0.2 µg/mL in Assay Buffer.
Prepare a curve of rmCystatin F (MW: 15249 Da) in Assay Buffer. Make the following serial dilutions: 6000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 5.208 nM.
Gently mix equal volumes of the rmCystatin L curve dilutions and the diluted active rhCathepsin L. Include a control (in duplicate) containing Assay Buffer and the diluted active rhCathepsin L.
Incubate mixtures at room temperature for 15 minutes.
Make a 5 fold dilution of the incubated curve dilutions in Assay Buffer.
Dilute Substrate to 20 µM in Assay Buffer.
Load 50 µL of the diluted incubated mixtures in a plate, and start the reaction by adding 50 µL of 20 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% inhibition concentration (IC₅₀) for rmCystatin F by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
The specific activity for rhCathepsin L at each point may be determined using the following formula (if needed):

Specific Activity (pmol/min/µg) =	Adjusted V_max* (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 L: 0.001 µg
rmCystatin F curve: 300, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.781 and 0.260 nM
Substrate: 10 µM

Reconstitution Calculator

Background: Cystatin F

Cystatin F, also known as leukocystatin and CMAP (Cystatin-like Metastasis-Associated Protein), is a new member of the Cystatin superfamily (1-3). Cystatin F is selectively expressed by hematopoietic cells and may be a biomarker for both liver metastasis and inflammatory lung disorders (3, 4). As a cysteine protease inhibitor, it shows selectivity towards Cathepsin L and legumain (1, 2, 5). Compared to other secreted Cystatins including C, D, E/M, S, SA and SN, which contain two intrachain disulfide bonds, Cystatin F has two extra Cys residues that may be involved in interchain disulfide bonds. rmCystatin F and rhCystatin F (Catalog # 1889‑PI) exhibit disulfide bond-linked dimer formation, which was also the case for an E. coli expressed fusion protein containing mature human Cystatin F and glutathione S-transferase (2).

References