Recombinant Human KLK3 (carrier-free)

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Other Names
Prostate-specific Antigen, PSA, Kallikrein 3
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Human_KLK3_RECOM_BA_092816
The activity of human KLK3 was measured with 1 mM of fluorogenic substrate, MeO-Suc-Arg-Pro-Tyr-AMC , in the presence of 0.48, 0.24, 0.12, 0.06, 0.03, 0.015 µg of activated KLK3. The activity of activated human KLK3 is > 150 pmole/µg/min. Alternatively, the activity can be measured with 1 mM of chromogenic substrate, MeO-Suc-Arg-Pro-Tyr-pNA. The activity is > 150 pmole/µg/min.
  • Human_KLK3_RECOM_BA_092816
    The activity of human KLK3 was measured with 1 mM of fluorogenic substrate, MeO-Suc-Arg-Pro-Tyr-AMC , in the presence of 0.48, 0.24, 0.12, 0.06, 0.03, 0.015 µg of activated KLK3. The activity of activated human KLK3 is > 150 pmole/µg/min. Alternatively, the activity can be measured with 1 mM of chromogenic substrate, MeO-Suc-Arg-Pro-Tyr-pNA. The activity is > 150 pmole/µg/min.
Cat # Size Price Quantity Avail. Save
761402 10 µg £84
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761404 25 µg £198
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761406 100 µg £400
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Description

Tissue Kallikreins (KLKs) are a subgroup of serine proteases having diverse physiological functions. These enzymes play important roles in a variety of complex processes including reproductive function, inflammation, skin homeostasis, blood clotting, fibrinolysis, and possibly cancer. Growing evidence suggests that many kallikreins are implicated in carcinogenesis, and some have potential as novel cancer and other disease biomarkers. Kallikrein 3 (KLK3), commonly known as prostate specific antigen (PSA), is a secreted protein which belongs to the peptidase S1 family and Kallikrein subfamily. The deduced amino acid sequence of human KLK3 consists of a signal peptide, a short pro region and a mature enzyme. KLK3 is a glycoprotein produced almost exclusively by the prostate gland. It is produced for the ejaculate where it liquifies the semen in the seminal coagulum. Several studies have demonstrated that KLK3 exerts antiangiogenic activity in vitro and in vivo. KLK3 has been shown to cleave components of the extracellular matrix (ECM), e.g., laminin and fibronectin, as well as unidentified proteins in the basement membrane preparation Matrigel. KLK2 and KLK3 have the most organ restricted expression profile of all KLKs; specifically, they are abundantly expressed in the luminal epithelium of the prostate. Serum level of KLK3, called PSA in the clinical setting, is useful in the diagnosis and monitoring of prostatic carcinoma.

Product Details
Technical Data Sheet (pdf)

Product Details

Reactivity
Human
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 (please contact technical support for concentration, or use our Lookup tool if you have a lot number.)
Please note, new lots of the 100 µg size will be lot-specific and may differ from previous lots that had a fixed concentration.
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 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
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

Related FAQs

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.

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.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?
Use formula Specific activity (Units/mg) = 10e6/ ED50 (ng/mL)
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.

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For research use only. Not for diagnostic use. Not for resale. BioLegend will not be held responsible for patent infringement or other violations that may occur with the use of our products.

 

*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/ordering#license). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products, reverse engineer functionally similar materials, or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.

 

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Toll-Free Phone: 1-877-Bio-Legend (246-5343) Phone: (858) 768-5800 Fax: (877) 455-9587

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