Cystatin M/CST6 Protein, Human (HEK293, His)

Cystatin M/CST6 protein is a potent inhibitor of cathepsin L, cathepsin L2 (cathepsin V) and Legumain, regulating important proteolytic processes. It actively participates in epidermal keratinization and affects the formation of the skin barrier. Cystatin M/CST6 Protein, Human (HEK293, His) is the recombinant human-derived Cystatin M/CST6 protein, expressed by HEK293 , with C-6*His labeled tag.

Cystatin M/CST6 Protein serves as a high-affinity inhibitor for cathepsin L, cathepsin L2 (cathepsin V), and legumain, highlighting its role in modulating key proteolytic processes. Beyond its inhibitory function, this protein is actively involved in the regulation of epidermal cornification, contributing to the intricate processes of skin barrier formation. Additionally, Cystatin M/CST6 plays a significant role in hair follicle morphogenesis and maintenance, further underscoring its importance in fundamental aspects of skin biology and homeostasis. The dual functionality of Cystatin M/CST6 in enzyme inhibition and the regulation of skin development emphasizes its multifaceted role in maintaining the integrity and function of cutaneous tissues.

Measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (HY-P1759B). The IC₅₀ value is 5.578 nM that incubate at 37°C for 10 minutes.

Materials
Assay buffer: 50 mM Tris, pH 7.0
Papain
Cystatin M/CST6 Protein, Human (HEK293, His) (HY-P70062)
Substrate: Z-FR-AMC (HY-P1759B), 10 mM stock in DMSO

Procedure
1. Pre-cool assay buffer on ice.
2. Dilute papain to 100 μg/mL using assay buffer containing 5 mM DTT.
3. Incubate at room temperature for 15 minutes to activate papain.
4. Prepare Human CST6 dilution curve using assay buffer. Perform the following serial dilutions: 2000, 500, 250, 125, 62.5, 31.25, 15.625, 5, 1, 0 nM.
5. Dilute activated papain to 2.05 μg/mL.
6. Mix equal volumes of the Human CST6 dilution curve with the diluted active papain.
7. Incubate the mixture at 37°C for 15 minutes.
9. Dilute the Human CST6 and papain mixture 5-fold with assay buffer.
10. Add 50 μL of diluted incubation mixture at each concentration to a black microplate well. Add 50 μL of 200 μM substrate to initiate the reaction.
11. Read for 5 minutes in kinetic mode at excitation and emission wavelengths of 380 nm and 460 nm, respectively.
12. Determine the 50% inhibitory concentration (IC₅₀) of Human CST6 by plotting the RFU/min versus concentration relationship using 4-PL fitting.

Human

HEK293

C-6*His

Q15828 (R29-M149)

1474  [NCBI]

Full Length of Mature Protein

RPQERMVGELRDLSPDDPQVQKAAQAAVASYNMGSNSIYYFRDTHIIKAQSQLVAGIKYFLTMEMGSTDCRKTRVTGDHVDLTTCPLAAGAQQEKLRCDFEVLVVPWQNSSQLLKHNCVQM

Approximately 14.0-19.0 kDa

≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder.

1.Lyophilized from a 0.22 μm filtered solution of 20 mM Tris-HCl, 10% maltose, 0.1% Tween 80, pH 9.0.
2.Lyophilized from a 0.22 μm filtered solution of PBS, pH 8.0.
Please refer to the lot-specific COA for specific buffer information.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.