Recombinant Human Kallikrein 3/PSA Protein, CF Summary
Ala18-Pro261, with a C-terminal 10-His tag
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CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Lyophilized from a 0.2 μm filtered solution in Tris, NaCl and CaCl2.|
|Reconstitution||Reconstitute at 200 μg/mL in 50 mM Tris, 10 mM CaCl2,150 mM NaCl and 0.05% (w/v) Brij-35, pH 7.5.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, 1 M NaCl, pH 8.0
- Recombinant Human Kallikrein 3/PSA (rhKLK3) (Catalog # 1344-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate: Suc-Arg-Pro-Tyr-pNa (AnaSpec, Catalog # 20586), 10 mM in deionized water
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhKLK3 to 200 µg/mL in Activation Buffer.
- Dilute Thermolysin to 2 µg/mL in Activation Buffer.
- Combine equal volumes of 200 µg/mL rhKLK3 and 2 µg/mL Thermolysin.
- Incubate at 37 °C for 5 minutes.
- Dilute 1,10 Phenanthroline to 20 mM in Assay Buffer.
- Stop Thermolysin activity by adding 1,10 Phenanthroline to a final concentration of 10 mM.
- Dilute activated rhKLK3 to 20 ng/µL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load 50 µL of the 20 ng/µL rhKLK3 in a plate and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 2 mM Substrate.
- Read at a wavelength of 405 nm (bottom read) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol|
|ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Using the extinction coefficient 8800 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD. Per Well:
- rhKLK3: 1 µg
- Substrate: 1 mM
Recombinant Human Kallikrein 3 (Catalog # 1344-SE) is measured by its ability to cleave the colorimetric peptide substrate, Succinyl-Arg-Pro-Tyr-p-Nitroanilide (Suc-RPY-pNA).
Background: Kallikrein 3/PSA
Kallikrein 3, commonly known as prostate specific antigen (PSA), is a serine protease of the human tissue Kallikrein gene family (1). PSA is synthesized in the ductal and acinar epithelium of the prostate gland and secreted into the seminal plasma in high concentrations (0.5 - 2 g/L) (2). A small portion of PSA “leaks” into the systemic circulation, the levels of which increase significantly (30-fold) from prostate cancer tissue than normal prostate tissue (3). PSA has become a well established tumor marker that aids the diagnosis, staging, and follow up of prostate cancer.
The deduced amino acid sequence of human PSA consists of a signal peptide, a short pro region and a mature/active enzyme. The pro-enzyme is activated, possibly by active Kallikreins 2, 4 or 15 in vivo (4). rhPSA is activated by thermolysin, a zinc protease. The active PSA cleaves several tyrosyl peptide bonds in semenogelins I and II, which are the major gel-forming proteins produced by the seminal vesicles (5). Several inhibitors including serpin A3/ alpha 1-antichymotrypsin (ACT) and alpha 2‑macroglobulin are known to form complexes with PSA.
- Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.
- Ward, A.M. et al. (2001) Ann. Clin. Biochem. 38:633.
- Jain, S. et al. (2002) Postgrad. Med. J. 78:646.
- LiLja H. (2003) Urology 62:27.
- Takayama, T.K. et al. (1997) J. Biol. Chem. 272:21582.
Citations for Recombinant Human Kallikrein 3/PSA Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 5
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Protein detection in blood with single-molecule imaging
Authors: CP Mao, SC Wang, YP Su, SH Tseng, L He, AA Wu, RBS Roden, J Xiao, CF Hung
Science Advances, 2021;7(33):.
Sample Types: Reference Standard
Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidases.
Authors: Mukai S, Fukushima T, Naka D, Tanaka H, Osada Y, Kataoka H
FEBS J., 2008;275(5):1003-17.
Sample Types: Protein
Applications: Enzyme Assay
Quantum dot-based protein micro- and nanoarrays for detection of prostate cancer biomarkers.
Authors: Gokarna A, Jin LH, Hwang JS, Cho YH, Lim YT, Chung BH, Youn SH, Choi DS, Lim JH
Sample Types: Recombinant Protein
Activation profiles and regulatory cascades of the human kallikrein-related peptidases.
Authors: Yoon H, Laxmikanthan G, Lee J, Blaber SI, Rodriguez A, Kogot JM, Scarisbrick IA, Blaber M
J. Biol. Chem., 2007;282(44):31852-64.
Sample Types: Peptide
Applications: Enzyme Assay
Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.
Authors: Kantyka T, Fischer J, Wu Z, Declercq W, Reiss K, Schroder J, Meyer-Hoffert U
Sample Types: Enzyme Substrate
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