Recombinant Mouse Active Heparanase/HPSE Protein, CF

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

Recombinant Mouse Active Heparanase/HPSE Protein, CF Summary

• Details of Functionality: Measured by its ability to release biotinylated heparan sulfate from Recombinant Human Syndecan‑4 (Catalog # 2918-SD). 20 ng rmHPSE digestion will result in >50% of OD reduction compared with the Negative Control.
• Source: Mouse myeloma cell line, NS0-derived mouse Heparanase/HPSE protein
  Asp28-Ile535, with an N-terminal 6-His tag
• Accession #: Q6YGZ1
• N-terminal Sequence: Glu146 and Lys150
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 43 kDa (50 kDa subunit) and 9 kDa (8 kDa subunit).
  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: 40-58 kDa (50 kDa subunit) and 8-12 kDa (8 kDa subunit), 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 Tris, NaCl and E64.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM Sodium Acetate, pH 5.0
  • Recombinant Mouse Heparanase/HPSE (rmHPSE) (Catalog # 9788-GH)
  • HPSE Substrate/ (Catalog # ES020)
  • Human Syndecan-4 DuoSet Kit (Catalog # DY2918)
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
    
    And the following materials that are routinely used in ELISA
  • Coating Buffer (Catalog # DY006)
  • Wash Buffer (25X) (Catalog # WA126)
  • Reagent Diluent (10X) (Catalog # DY995)
  • Substrate Reagent Pack (Catalog # DY999)
  • Stop Solution (Catalog # DY994)
  1. Prepare an ELISA plate by following the DuoSet kit protocol.
  2. Dilution factor determination:
     a. Dilute the HPSE Substrate stock 100-fold in Reagent Diluent (this will be the first dilution point).
     b. Further prepare a 2-fold serial dilution series of the above diluted HPSE Substrate with Reagent Diluent for 6 points.
     c. Load 100 µL of each point onto the prepared ELISA plate in duplicate. Load 100 µL of Reagent Diluent to 2 separate wells for blank control.
     d. Cover the plate and incubate at room temperature for 2 hours.
     e. Follow the DuoSet Assay Procedure from step 4 to step 9 to complete the assay.
     f. Determine the dilution factor (n) that achieves an OD between 1.8-3.0.
  3. rmHPSE Activity Detection:
     a. Dilute the HPSE Substrate stock by n/10-fold in Assay Buffer.
     b. Combine 10 µL of rmHPSE with 10 µL of the diluted HPSE Substrate in a vial. For negative control, combine 10 µL of Assay Buffer and 10 µL of the diluted HPSE Substrate in a vial.
     c. Incubate reactions and negative control at 37 °C for 2 hours.
     d. After incubation, heat all reactions and negative control at 95 °C for 2 minutes to inactivate rmHPSE.
     e. Add 220 µL of Reagent Diluent to each reaction and negative control. Mix well.
     f. Load 100 µL of each sample onto the prepared ELISA plate in duplicate.
     g. Cover the plate and incubate for 2 hours at room temperature.
     h. Follow the DuoSet Assay Procedure from step 4 to step 9 to complete the assay.
  4. Calculate % OD reduction compared with the negative control:
     
     [1-(OD of the wells of rmHPSE treated/OD of the wells of negative control)] x 100 = % OD reduction
  Per Reaction:
  • rmHPSE: 20 ng

Notes

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

Alternate Names for Recombinant Mouse Active Heparanase/HPSE Protein, CF

• EC 3.2
• Endo-glucoronidase
• HEP
• Heparanase
• Heparanase-1
• HPA1
• HPAheparanase exon 9 and 10 deletion
• HPR1
• HPSE
• HPSE1heparanase-1
• HSE1
• HSE1heparanase exon10-deletion

Background

Heparanase (HPSE) selectively cleaves heparan sulfate (HS) at specific sites on HS proteoglycans (HSPGs) (1, 2, 3, 4). The enzyme is synthesized as an inactive 65 kDa proenzyme that is secreted via the Golgi apparatus and associates with the cell membrane through interaction with HSPGs (5). It is then endocytosed and transferred to lysosomes (6) where cathepsin L activates it by removing an internal inhibitory peptide, forming a heterodimer composed of an 8 kDa and a 50 kDa subunit (7, 8). Under certain stimuli, the active enzyme is transferred back to the cell surface, where it participates in extracellular matrix degradation and remodeling (9). HPSE facilitates cell migration associated with metastasis, wound healing and inflammation (10). An increase in its activity is associated with an increase in VEGF activity, which further enhances angiogenesis (11). HPSE also enhances shedding of syndecans and increases endothelial invasion and angiogenesis in myelomas (12). It acts as a procoagulant by increasing the generation of activation factor X in the presence of tissue factor and activation factor VII (13). In addition, it increases cell adhesion to the extracellular matrix (ECM), independent of its enzymatic activity (14). HPSE is highly expressed in placenta and spleen and weakly expressed in lymph node, thymus, peripheral blood leukocytes, bone marrow, endothelial cells, fetal liver and tumor tissues (15). Mouse HPSE shows 76% identity to human HPSE at amino acid sequence. The enzyme activity of recombinant mouse HPSE was assayed using recombinant syndecan 4 that was biotinylated at the non-reducing end of its HS chains (catalogue ES020) in ELISA format (16).

Bioinformatics

• Uniprot:
  • Q6YGZ1()