Recombinant Human Cathepsin L (carrier-free)

Product Details

Source

Human Cathepsin L, amino acid (Thr18-Val333) (Accession: P07711), with a C-terminal 8His tag, was expressed in 293E cells.

Molecular Mass

The 327 amino acid recombinant protein has a predicted molecular mass of approximately 25.5 kD. The protein migrates at approximately 31 kD and 27 kD by SDS-PAGE in DTT-reduced and non-reduced conditions, respectively. The predicted N-terminal amino acid is Thr.

Purity

> 96%, as determined by Coomassie stained SDS-PAGE

Formulation

0.22 µm filtered protein solution is in 50 mM sodium acetate, 0.1% glacial acetic acid. 0.1 M NaCl, 40% glycerol, pH 5.

Endotoxin Level

Less than 0.1 EU per µg cytokine as determined by the LAL method

Concentration

Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)

Storage & Handling

Unopened vial can be stored at -20°C or -70°C for six months. For maximum results, quick spin vial prior to opening. Avoid repeated freeze/thaw cycles.

Activity

Recombinant human Cathepsin L is measured by its ability to cleave Z-LR-AMC. Cleavage product is detected by an increase in fluorescence at excitation and emission at 380 and 460 nm, respectively. The recommended specific activity is > 23,000 pmol/min/µg.

Application

Bioassay

Application Notes

Human Cathepsin L Assay Procedure
 

Human Cathepsin L activity is measured by its ability to cleave the fluorogenic peptide substrate Z-LR-AMC. The accumulation of cleavage product is quantified by the increase in fluorescence at 460 nm upon excitation at 380 nm.
 

Materials and Buffers
 

1. Assay Buffer: 50 mM MES, 5 mM DTT, 1 mM EDTA, 0.005% (w/v) Brij-35, pH 6.0

2. Human CTSL substrate: Z-LR-AMC (Bachem Cat. No. I-1960)

3. Recombinant human cathepsin L

4. F16 Black Maxisorp Plate (Nunc, Cat. No. 475515)

5. Fluorescent Plate Reader (Model: SpectraMax Molecular Devices) or equivalent
 

Assay Procedure
 

1. Dilute the human CTSL to 40 µg/mL in assay buffer.

2. Incubate the diluted human CTSL on ice for 15 minutes.

3. Dilute the human CTSL to 0.08 ng/µL in assay buffer. Make six 1:2 dilutions in a black well plate. Include a blank with 50 µL assay buffer.

4. Dilute the substrate to 80 µM in assay buffer.

5. Start the reaction by adding 50 µL of the diluted substrate to each well. 

6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.

7. 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 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).
 

Per well:

• Human CTSL: 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 ng

• Substrate: 40 µM

Additional Product Notes

BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are validated in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.

 

Antigen Details

Structure

Enzyme endopeptidase

Distribution

Ubiquitously expressed endopeptidase, highly expressed in macrophages

Function

Intracellular protein catabolism, participates in autophagy, controls release of trypsinogen from pancreatic cells and activation in duodenum, regulates secretory vesicles in the neuroendocrine system, homeostasis is cardiomyocytes. Activated and mature cathepsin L are regulated by cystatins, endogenous inhibitors.

Interaction

Substrates LC3-II, GABARAP-II, GTPase dynamin, and synaptopodin.

Ligand/Receptor

Peptide bonds with aromatic amino acids (aa) in P2 and hydrophobic aa in P3 position

Bioactivity

Recombinant Human Cathepsin L cleaves fluorogenic peptide substrate Z-LR-AMC.

Cell Type

Endothelial cells, Epithelial cells, Fibroblasts, Macrophages, Monocytes, T cells

Biology Area

Cancer Biomarkers, Cardiovascular Biology, Cell Biology, Cell Proliferation and Viability, COVID-19, Immuno-Oncology, Neuroscience

Molecular Family

Enzymes and Regulators, Lysosomal Markers, Proteases

Antigen References

1. Creasy BM & McCoy KL. 2011. Cell Immunol. 267:56.
2. Coulombe R, et al. 1996. EMBO J. 15:5492.
3. Sudhan DR & Siemann DW, 2015. Pharmacol Ther. 155:105.
4. Katopodis, P, et al. 2020. Int J Oncol. 57:533.
5. Hopkins JN, et al. 2018. J Virol. 92:e01179.
6. Ueno T. & Takahashi K. 2009. Autophagy. 5:878
7. Dana D & Pathak SK. 2020. Molecules 25:698.

Gene ID

1514 View all products for this Gene ID

UniProt

View information about Cathepsin L on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

How does the activity of your recombinant proteins compare to competitors?

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

What is the specific activity or ED50 of my recombinant protein?

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Have your recombinants been tested for stability?

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Does specific activity of a recombinant protein vary between lots?

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)

Certificate of Analysis