Source	Mouse myeloma cell line, NS0-derived human Cathepsin D protein Leu21-Leu412, with a C-terminal 10-His tag
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.
N-terminal sequence Analysis	Leu21
Predicted Molecular Mass	44 kDa
SDS-PAGE	50 kDa, reducing conditions
Activity	Measured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH₂ (Catalog # ES001). The specific activity is >350 pmol/min/µg, as measured under the described conditions.

Formulation, Preparation and Storage

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.

1014-AS

Formulation	Supplied as a 0.2 μm filtered solution in MES 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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Assay Procedure

Materials

Assay Buffer: 0.1 M NaOAc, 0.2 M NaCl, pH 3.5
Recombinant Human Cathepsin D (rhCathepsin D) (Catalog # 1014-AS)
Substrate: MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH₂ (Catalog # ES001), 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 D to 20 µg/mL in Assay Buffer.
Aliquot 50 µL of 20 µg/mL rhCathepsin D.
Incubate at 37 °C for 30 minutes.
Dilute incubated rhCathepsin D to 1 ng/µL in Assay Buffer.
Dilute Substrate to 60 µM in Assay Buffer.
Load 50 µL of 1 ng/µL rhCathepsin D in a plate, and start the reaction by adding 50 µL of 60 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 60 µM Substrate.
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 V_max* (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).

Per Well:

rhCathepsin D: 0.050 µg
Substrate: 30 µM

Background: Cathepsin D

Cathepsin D is a lysosomal aspartic protease of the pepsin family (1). Human cathepsin D is synthesized as a precursor protein, consisting of a signal peptide (residues 1‑18), a propeptide (residues 19‑64), and a mature chain (residues 65‑412) (2‑4). The mature chain can be processed further to the light (residues 65‑161) and heavy (residues 169‑412) chains. It is expressed in most cells and overexpressed in breast cancer cells (5). It is a major enzyme in protein degradation in lysosomes, and also involved in the presentation of antigenic peptides. Mice deficient in this enzyme showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound neuronal ceroid lipofucinosis, indicating that cathepsin D is essential for proteolysis of proteins regulating cell growth and tissue homeostasis (6). Cathepsin D secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogeneous inhibitor of angiogenesis (6).

Item	Value
References	Conner et al. in Handbook of Proteolytic Enzymes Barrett (1998) Academic Press, San Diego, p. 828. Faust, et al. (1985) Proc. Natl. Acad. Sci. USA 82:4910. Westley and May (1987) Nucl. Acid Res. 15:3773. Redecker, et al. (1991) DNA Cell Biol. 10:423. Rochefort, et al. (2000) Clin. Chim. Acta. 291:157. Tsukuba, et al. (2000) Mol. Cells 10:601.
Entrez Gene IDs	1509 (Human); 13033 (Mouse)
Alternate Names	CPSD, CTSD, Cathepsin D, EC 3.4.23, EC 3.4.23.5, MGC2311, cathepsin D (lysosomal aspartyl protease), lysosomal aspartyl peptidase, lysosomal aspartyl protease, neuronal 10

Citations (8)

Showing 1 - 8 of 8 citations Showing All

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Citations are publications that use Bio-Techne products. Selected citations for Recombinant Human Cathepsin D Protein, CF include:

Applications: Bioassay

SL Ivry et al. (2017), Global protease activity profiling provides differential diagnosis of pancreatic cysts Clin. Cancer Res., 0(0):.
PMID: 28424202
Species: N/A
Sample Types: Protein
Applications: Bioassay

YJ Hsieh et al. (2017), Oxidation of protein-bound methionine in Photofrin-photodynamic therapy-treated human tumor cells explored by methionine-containing peptide enrichment and quantitative proteomics approach Sci Rep, 7(1):1370.
PMID: 28465586
Species: Human
Sample Types: Protein
Applications: Bioassay

CW Lee et al. (2017), The lysosomal protein cathepsin L is a progranulin protease Mol Neurodegener, 12(1):55.
PMID: 28743268
Species: Human
Sample Types: Protein
Applications: Enzyme Assay

Khalkhali-Ellis et al. (2014), Cleavage of Histone 3 by Cathepsin D in the involuting mammary gland. PLoS ONE, 9(7):e103230.
PMID: 25054204
Species: Human
Sample Types: Whole Cells
Applications: Bioassay

Derocq D et al. (2012), Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis. Oncogene, 31(26):3202-12.
PMID: 22081071
Species: Human
Sample Types: Protein
Applications: Enzyme Assay

Zou F et al. (2011), Regulation of cathepsin g reduces the activation of proinsulin-reactive T cells from type 1 diabetes patients. PLoS ONE, 6(8):e22815.
PMID: 21850236
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay

Ahmed RR et al. (2010), BACE1 and BACE2 enzymatic activities in Alzheimer's disease. J. Neurochem., 112(4):1045-53.
PMID: 19968762
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay

Cheng T et al. (2006), Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification. J. Biol. Chem., 281(23):15893-9.
PMID: 16565075

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Application: Immunoassay Standard

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