Recombinant Human KLK3 (carrier-free)

Product Details

Source

Human KLK3, amino acids Ala18-Pro261(Accession# NM_001648) with a C-terminal TG-8H-GGQ tag was expressed in CHO cells.

Molecular Mass

The 257 amino acid recombinant protein has a predicted molecular mass of approximately 28.3 kD. The DTT-reduced and non reduced proteins migrates at approximately 30 kD and 28 kD respectively by SDS-PAGE.

Purity

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

Formulation

0.22 µm filtered protein solution is in TCN (25 mM TRIS, 10 mM CaCl2, 150 mM NaCl, pH 7.5).

Endotoxin Level

Less than 1 EU per µg protein as determined by the LAL method.

Concentration

10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. 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

Human KLK3 cleaves peptide substrate MeO-Suc-Arg-Pro-Tyr-pNA and MeO-Suc-Arg-Pro-Tyr-AMC with a specific activity value > 150 pmol/µg/min.

Recommended Usage

Bioassay

Application Notes

Human KLK-3 Activity Assay with fluorogenic substrate

Human KLK-3 activity is measured by its ability to cleave a fluorogenic peptide substrate Suc-Arg-Pro-Tyr-AMC after hKLK-3 is activated by thermolysin. The progress of the substrate cleavage is monitored by increase in intensity of fluorescence emission at 460 nm with excitation at 380 nm.

Materials and Buffers

1. Activation Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35.
2. Assay Buffer: pH 8.0, 50 mM Tris, 1.0 M NaCl.
3. 500 mM EDTA solution at pH 8.0 for stopping the activation.
4. Bacterial Thermolysin (TLN) (SIGMA, Cat # P1512).
5. Human KLK-3: BioLegend
6. KLK-3 substrate:  Suc-Arg-Pro-Tyr-AMC

Assay Procedures

1. Dilute TLN to 0.5 µg/mL in Activation Buffer.
2. Dilute hKLK-3 to 200 µg/mL in Activation Buffer.
3. Combine equal volumes of 200 µg/mL of hKLK-3 and 0.5 µg/mL TLN to make the activating concentration of 100 µg/mL of hKLK7 and 0.25 µg/mL of TLN.
4. Incubate at room temperature for 1 hour.
5. Stop the reaction with equal volume of 500 mM EDTA at pH 8.0. At this moment, hKLK-3 is at 50 µg/mL.
6. Dilute the activated hKLK-3 to 4 µg/mL with Assay Buffer.
7. Dilute the substrate stock at 2 mM in Assay Buffer.
8. Load 50 µL of the activated hKLK-3 in 4 µg/mL into a plate, and start a reaction by adding 50 µL of 2 mM of the substrate solution into each well.
9. Read the reaction progress by monitoring fluorescence emission at 460 nm with excitation at 380 nm in kinetic mode for 5 minutes.
10. The final human KLK-3 concentration is at 2 µg/mL (0.2 µg), the substrate concentration at 1 mM, TLN at 0.005 µg/mL, and EDTA at 10 mM.

Conversion factor of AMC is 0.3 pmol/RFU

Human KLK-3 Activity Assay with chromogenic substrate

Human KLK-3 activity is measured by its ability to cleave a fluorogenic peptide substrate Suc-Arg-Pro-Tyr-pNA after hKLK-3 is activated by thermolysin. The progress of the substrate cleavage is monitored by increase in intensity of absorbance at 405 nm.

Materials and Buffers

1. Activation Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35.
2. Assay Buffer: pH 8.0, 50 mM Tris, 1.0 M NaCl.
3. 500 mM EDTA solution at pH 8.0 for stopping the activation.
4. Bacterial Thermolysin (TLN) (SIGMA, Cat # P1512).
5. Human KLK-3: BioLegend
6. KLK-3 substrate:  Suc-Arg-Pro-Tyr-pNA

Assay Procedures

1. Dilute the TLN to 0.5 µg/mL in Activation Buffer.
2. Combine equal volumes of provided 200 µg/mL of hKLK-3 and 0.5 µg/mL TLN to make activating concentration of 100 µg/mL of hKLK7 and 0.25 µg/mL of TLN.
3. Incubate at room temperature for 1 hour.
4. Stop the reaction with equal volume of 500 mM EDTA at pH 8.0. At this moment, hKLK-3 is at 50 µg/mL.
5. Dilute the activated hKLK-3 to 10 µg/mL with Assay Buffer.
6. Dilute the substrate stock at 2 mM in Assay Buffer.
7. Load 50 µL of the activated hKLK-3 in 10 µg/mL into a plate, and start a reaction by adding 50 µL of 2 mM of the substrate solution into each well.
8. Read the reaction progress by monitoring absorbance at 405 nm in kinetic mode for 5 minutes.
9. The final human KLK-3 concentration is at 5 µg/mL (0.5 µg), the substrate concentration at 1 mM, TLN at 0.0125 µg/mL (1.25 ng), and EDTA at 25 mM.

Conversion factor of pNA at pH 8.0 by using extinction coefficient 8800 M^-1cm^-1, 0.32 cm of light path correction, and total volume of 100 µL: 35.5 pmol/mAu.

This protein is in the latent form and needs to be activated for bioassay.

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

Ductal and acinar epithelium of the prostate gland.

Function

It liquifies the semen in the seminal coagulum; Serpin A3 (alph-1-antichymotrypsin) and alpha-2-macroglobulin are known inhibitors.

Interaction

Semenogelin I and II; Serpin A3.

Ligand/Receptor

Serpin A3 (Alpha-1-antichymotrypsin) and alpha-2-macroglobulin.

Bioactivity

Degrades semenogelin I an II; also ECM components.

Biology Area

Stem Cells

Molecular Family

Enzymes and Regulators

Antigen References

1. Takayama TK, et al. 1997. J. Biol. Chem. 272:21582.
2. LiLja H. 2003. Urology. 62:27.
3. Emami N, Diamandis EP. 2008. Clin. Chem. 54:1600.
4. Shaw JL, Diamandis EP. 2007. Clin. Chem. 53:1423.
5. Thorek DL, et al. 2013. Thromb. Haemost. 110:484.
6. Hong SK. 2014. Biomed. Res. Int. 2014:526341.

Gene ID

354 View all products for this Gene ID

UniProt

View information about KLK3 on UniProt.org

Documentation

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