Cathepsin D Protein, Human (HEK293, His, solution)

Cathepsin D Protein, Human (HEK293, His, solution) is an approximately 50.0 kDa cathepsin D protein with a His-flag. Cathepsin D is a representative lysosomal aspartic proteinases and belongs to the peptidase C1 family^[1].

Cathepsin D (CTSD), a well-known lysosomal aspartyl protease and belongs to the peptidase C1 family, which is a normal and major component of lysosomes, and is found in almost all cells and tissues of mammals^[1].
Human cathepsin D is synthesized as a precursor protein, consisting of a signal peptide (residues 1-18), a propeptide (residues 1964), and a mature chain (residues (65412))^[2].

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH2. The specific activity is >600 pmol/min/µg, as measured under the described conditions.

Materials
Detection buffer: 0.1 M NaOAc, 0.2 M NaCl, pH 3.5
Cathepsin D Protein, Human (HEK293, His, solution) (HY-P7748)
Substrate: MOCAc-PLGL(Dpa)AR (HY-131498), 2 mM dissolved in DMSO
Standard: MCA-Pro-Leu-OH

Procedure
1. Standard Curve Preparation: Dilute the standard with the detection buffer to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 μmol/L. Add 100 μL of each standard concentration to a 96-well plate. Read the plate in kinetic mode for 5 minutes with excitation and emission wavelengths set at 320 nm and 405 nm (top read), respectively. Plot the standard concentrations on the x-axis and the measured RFU (Relative Fluorescence Units) on the y-axis to generate the standard curve and obtain the standard curve equation.
2. Dilute the protein sample to 20 μg/mL with the detection buffer.
3. Incubate at 37℃ for 30 minutes.
4. Dilute the incubated protein sample to 1 μg/mL with the detection buffer.
5. Dilute the substrate to 60 μM with the detection buffer.
6. Add 50 μL of the diluted protein sample to the 96-well plate and initiate the reaction by adding 50 μL of 60 μM substrate. For the blank wells, add 50 μL of detection buffer and 50 μL of 60 μM substrate.
7. Read the plate in kinetic mode for 5 minutes with excitation and emission wavelengths set at 320 nm and 405 nm, respectively.
8. Calculate the specific activity:

Human

HEK293

C-6*His

P07339 (L21-L412)

1509  [NCBI]

Full Length of Mature Protein

LVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVPAVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWIHHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFGEATKQPGITFIAAKFDGILGMAYPRISVNNVLPVFDNLMQQKLVDQNIFSFYLSRDPDAQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAIVDTGTSLMVGPVDEVRELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQAGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARLHHHHHH

Approximately 50.0 kDa

Greater than 95% as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filter solution of 20 mM MES, 150 mM NaCl, pH 5.5.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. T Tsukuba, et al. New functional aspects of cathepsin D and cathepsin E. Mol Cells. 2000 Dec 31;10(6):601-11.  [Content Brief]

  [2]. P L Faust, et al. Cloning and sequence analysis of cDNA for human cathepsin D. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4910-4.  [Content Brief]

  [3]. B Redecker, et al. Molecular organization of the human cathepsin D gene. DNA Cell Biol  [Content Brief]

  [4]. T Tsukuba, et al. New functional aspects of cathepsin D and cathepsin E. Mol Cells  [Content Brief]