Recombinant Human Kallikrein 12 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Kallikrein 12 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate Boc-VPR-AMC (Catalog # ES011). The specific activity is > 4,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Kallikrein 12 protein
  Ala18-Asn248, with a C-terminal 10-His tag
• Accession #: NP_665901
• N-terminal Sequence: Ala18
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: KLK12
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Theoretical MW: 26 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 37 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 100 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 8.0
  • Assay Buffer: 100 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% (w/v) Brij-35, pH 7.5
  • Recombinant Human Kallikrein 12 (rhKLK12) (Catalog # 3095-SE)
  • Fluorescent Peptide Substrate: BOC-Val-Pro-Arg-AMC (Catalog # ES011), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhKLK12 to 100 µg/mL in Activation Buffer.
  2. Incubate 100 µg/mL rhKLK12 at 37 °C for 24 hours.
  3. Dilute rhKLK12 to 0.2 ng/µL in Assay Buffer.
  4. Dilute Substrate to 200 µM in Assay Buffer.
  5. Load into plate 50 µL of 0.2 ng/µL rhKLK12, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing Assay Buffer in place of rhKLK12.
  6. Read plate 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 Vmax* (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:
  • rhKLK12: 0.01 µg
  • Substrate: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Kallikrein 12 Protein, CF

• DKFZp686H1078
• EC 3.4.21
• EC 3.4.21.-
• Kallikrein 12
• Kallikrein-like protein 5
• kallikrein-related peptidase 12
• KLK12
• KLK-L5
• KLKL5kallikrein-12
• KLK-L5MGC42603

Background

Human tissue Kallikrein 12, encoded by the KLK12 gene, is a secreted serine protease that belongs to the human tissue kallikrein family. It is present in many tissues, such as salivary gland, stomach and breast. KLK12 displays trypsin-like enzymatic activity. This activity can be inhibited by Serpin F2 (Catalog # 1470-PI). The physiological functions of KLK12 still remain unclear. Its expression is modulated by steroid hormones and is down‑regulated in breast cancer (2). Human KLK12 has three splice variants resulting from alternative splicing of the 3’ end. The KLK12 produced by the R&D Systems corresponds to the full-length classical form, also known as isoform 2 (1). The amino acid sequence of human KLK12 is 80%, 77%, 71% and 66% to that of bovine, canine, mouse, and rat. The recombinant human KLK12 can be autoactivated under the conditions described in the Activity Assay Protocol. The active enzyme has the N-terminal sequence of I₂₂FNGTECGRNS.

1. Yousef, G.M. et al. (2000) Genomics. 69:331.
2. Yousef, G.M. and E.P. Diamandis (2001) Endocrine Rev. 22:184.

Publications for Kallikrein 12 (3095-SE)(6)

Publications using 3095-SE

• MR Straus, GR Whittaker A peptide-based approach to evaluate the adaptability of influenza A virus to humans based on its hemagglutinin proteolytic cleavage site PLoS ONE, 2017;12(3):e0174827. 2017 [PMID: 28358853] (EnzAct, Influenza A Virus)
• Kryza T, Achard C, Parent C, Marchand-Adam S, Guillon-Munos A, Iochmann S, Korkmaz B, Respaud R, Courty Y, Heuze-Vourc'h N Angiogenesis stimulated by human kallikrein-related peptidase 12 acting via a platelet-derived growth factor B-dependent paracrine pathway. FASEB J, 2014;28(2):740-51. 2014 [PMID: 24225148]
• Guillon-Munos A, Oikonomopoulou K, Michel N, Smith CR, Petit-Courty A, Canepa S, Reverdiau P, Heuzé-Vourc'h N, Diamandis EP, Courty Y Kallikrein-related peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors. J. Biol. Chem., 2011;286(29):25505-18. 2011 [PMID: 21628462] (EnzAct, Human)
• Sexton DJ, Chen T, Martik D, Kuzmic P, Kuang G, Chen J, Nixon AE, Zuraw BL, Forteza RM, Abraham WM, Wood CR Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases. Biochem. J., 2009;422(2):383-92. 2009 [PMID: 19527222] (Surface Plasmon Resonance, Human)
• Yoon H, Laxmikanthan G, Lee J, Blaber SI, Rodriguez A, Kogot JM, Scarisbrick IA, Blaber M Activation profiles and regulatory cascades of the human kallikrein-related peptidases. J. Biol. Chem., 2007;282(44):31852-64. 2007 [PMID: 17823117] (EnzAct, N/A)
• Memari&lt;/LastName&gt;&lt;ForeNam N&lt;/Initial, Memari N, Jiang W, Diamandis EP, Luo LY Enzymatic properties of human kallikrein-related peptidase 12 (KLK12). Biol. Chem., 2007;388(4):427-35. 2007 [PMID: 17391064] (EnzAct, Human)

Bioinformatics

• Gene Symbol: KLK12
• Entrez:
  • 43849(Human)
• Uniprot:
  • NP_665901()