Recombinant Human KLK5 (carrier-free)

Product Details

Source

Human KLK5, amino acid (Ile67- Ser293) (Accession: # Q9Y337), with a C-terminal GGS-8H tag was expressed in CHO cells.

Molecular Mass

The 236 amino acid recombinant protein has a predicted molecular mass of approximately 26.1 kD. The DTT-reduced and non-reduced protein migrates at approximately 60 and 50 kD by SDS-PAGE respectively. The predicted N-terminal amino acid is Ile.

Purity

> 95%, as determined by Coomassie stained SDS-PAGE

Formulation

0.22 µm filtered protein solution is in 25 mM MES, 150 mM NaCl, 20% glycerol pH 5.5

Endotoxin Level

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

Concentration

10 and 25 µg sizes are bottled at 200 µg/mL.

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 KLK5 activity is measured by its ability to cleave the fluorogenic peptide substrate Boc-Val-Pro-Arg-AMC. The specific activity is > 200 pmol/min/µg in the presence of 0.1 µg of recombinant human KLK5.

Application

Bioassay

Application Notes

Human KLK5 Activity Assay

Recombinant Human KLK5 activity is measured by its ability to cleave the fluorogenic peptide substrate Boc-Val-Pro-Arg-AMC. The increase of the product is monitored by an increase in the intensity of fluorescence at 460 nm with excitation at 380 nm. Perform assay rapidly. The specific activity is > 200 pmol/min/µg in the presence of 0.1 µg of recombinant human KLK5.

Materials

• Assay buffer: 50 mM Tris, 0.005% (w/v) Brij-35, pH 8.0
• Recombinant human KLK5
• Fluorogenic peptide substrate: Boc-Val-Pro-Arg-AMC
• F16 Black Maxisorp Plate (Nunc, Cat. No. 475515)

Activity Assay Procedures

1. Dilute the substrate in assay buffer at 200 µM. Wrap the substrate container in aluminum foil.
2. In assay buffer, prepare 50 µL of serial dilutions of human KLK5. Start at a concentration of 16 µg/mL and dilute 1 to 2 six times after.
3. Add 50 µL of 200 µM substrate to wells with 50 µL of prepared dilutions.
4. Include a substrate black with 50 µL of assay buffer and 50 µL of 200 µM substrate without any human KLK5.
5. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes.
6. Calculate specific activity using the following equation:

Specific Activity (pmol/min/µg)=  ((Adjusted V_max* (RFU/min) x Conversion Factor** (pmol/RFU))
                                                                                                  amount of enzyme (µg)

Final assay conditions per well:
hKLK5: 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 µg
Substrate: 100 µM

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 verified 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

Monomer

Distribution

Highly expressed in endocrine or hormone-responsive tissues, ovary, testis, breast, and skin (epidermis). Secreted by T cells and neutrophils.

Function

Involved in skin desquamation, skin physiology. Upregulated by estrogens and progestins. Inhibited by serpins alpha(2)-antiplasmin and antithrombin. Inhibited by LEKTI fragments.

Interaction

DPP4 (CD26)

Ligand/Receptor

Substrates: plasminogen, kininogen, fibrinogen, vitronectin, laminin, and collagens type I, II, III, IV

Bioactivity

Human KLK5 activity is measured by its ability to cleave fluorogenic peptide substrate Boc-Val-Pro-Arg-AMC.

Cell Type

Neutrophils, T cells, Th17

Biology Area

Cancer Biomarkers, Immuno-Oncology

Molecular Family

Enzymes and Regulators, Proteases

Antigen References

1. Brattsand M & Egelrud T. 1999. J Biol Chem. 274:30033-40.
2. Komatsu N, et al. 2003. J Invest Dermatol. 121:542-9.
3. Yousef GM, et al. 2004. Br J Cancer. 90:167-72.
4. Michael IP, et al. 2005. J Biol Chem. 280:14628-35.
5. Lizama AJ, et al. 2015. Innate Immun. 6:575-86.
6. Nargis T, et al. 2017. Mol Metab. 6:1529-1539.

Gene ID

25818 View all products for this Gene ID

UniProt

View information about KLK5 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