Recombinant Human Cathepsin X/Z/P Protein, CF Summary
Ala21-Val303 (Gly23Val & Ser48Thr), with a C-terminal 10-His tag
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 Sodium Acetate and NaCl.|
|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.
- Activation Buffer: 25 mM Sodium Acetate, pH 3.5
- Assay Buffer: 25 mM Sodium Acetate, 5 mM Dithiothreitol (DTT), pH 3.5
- Dithiothreitol (DTT), 1 M stock in deionized water
- Recombinant Human Cathepsin X/Z/P (rhCathepsin X/Z/P) (Catalog # 934-CY)
- Substrate: MCA-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(DNP)-OH (Catalog # ES005)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rhCathepsin X/Z/P.
- Dilute rhCathepsin X/Z/P to 20 µg/mL in Activation Buffer.
- Dilute DTT stock to 10 mM in Activation Buffer.
- Mix equal volumes of 20 µg/mL rhCathepsin X/Z/P and 10 mM DTT.
- Incubate at room temperature for 5 minutes.
- Dilute activated rhCathepsin X/Z/P to 0.4 ng/µL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the 0.4 ng/µL rhCathepsin X/Z/P in a black well plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate without any rhCathepsin X/Z/P.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
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-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhCathepsin X/Z/P: 0.02 µg
- Substrate: 10 µM
Background: Cathepsin X/Z/P
Cathepsin X (also known as Cathepsin Z and P) is a cysteine protease of the papain family (1-5). Compared to other members of the papain family, Cathepsin X has a short proregion and unique insertions. The cysteine residue in the proregion forms a covalent and reversible bond with the active site cysteine residue (6). Acting as a carboxypeptidase, Cathepsin X displays a unique specificity (7-10). It is ubiquitously expressed in human tissues and conserved in other species such as mouse, nematode and echiuran. The nematode enzyme is apparently involved in molting of third stage larvae (11).
- Nagler, D.K. and R. Menard (1998) FEBS Lett. 434:135.
- Santamaria, I. et al. (1998) J. Biol. Chem. 273:16816.
- Deussing, J. et al. (2000) Biochim. Biophys. Acta 1491:93.
- Pungercar, J. and G. Ivanovski (2000) Pflugers Arch. Eur. J. Physiol. 439:R116.
- Pungercar, J. et al. (2000) Pflugers Arch. Eur. J. Physiol. 439:R119.
- Sivaraman, J. et al. (2000) J. Mol. Biol. 295:935.
- Menard, R. et al. (2001) Biol. Chem. 382:839.
- Therrien, C. et al. (2001) Biochemistry 40:2702.
- Klemencic, I. et al. (2000) Eur. J. Biochem. 267:5404.
- Guncar, G. et al. (2000) Structure Fold Des. 8:305.
- Lustigman, S. et al. (1996) J. Biol. Chem. 271:30181.
Citations for Recombinant Human Cathepsin X/Z/P 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.
Citations: Showing 1 - 2
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Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions.
Authors: Burster T, Marin-Esteban V, Boehm BO, Dunn S, Rotzschke O, Falk K, Weber E, Verhelst SH, Kalbacher H, Driessen C
Biochem. Pharmacol., 2007;74(10):1514-23.
Sample Types: Cell Lysates
Applications: Enzyme Assay
An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells.
Authors: Falgueyret JP, Black WC, Cromlish W, Desmarais S, Lamontagne S, Mellon C, Riendeau D, Rodan S, Tawa P, Wesolowski G, Bass KE, Venkatraman S, Percival MD
Anal. Biochem., 2004;335(2):218-27.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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Fluorogenic Peptide Substrates
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