Heparanase is an enzyme that cleaves heparan sulfate, and thereby releases growth factors that are implicated in tumor cell proliferation, metastasis and angiogenesis. Cisbio Bioassays' HTRF®-based assay monitors heparanase activity using a proprietary heparan sulfate substrate labeled with both biotin and Eu3+ Cryptate. This homogeneous assay has the robustness of HTRF technology and rapidly produces high quality results.
The HTRF heparanase assay is based on our proprietary heparan sulfate substrate, labeled with both biotin and Eu3+ Cryptate. Active heparanase enzyme cleaves the substrate causing the loss of energy transfer, and thus a reduction in SA-XL665 emissions.
The assay is run as a two-step process:
Stimulation step: heparanase enzymatic reaction (e.g. 30 min at 37°C, to be optimized)
Detection step: Heparanase activity is detected by the addition of streptavidin-XL665 (SA-XL665).
The effects of heparanase enzyme concentration and the enzyme reaction time on substrate degradation are shown here. Based on the linearity of the enzyme dose-response, enzyme consumption and incubation time results, 90 ng/mL of heparanase incubated for 30 minutes at 37ºC was chosen for inhibitor screening.
HTRF Heparanase assay was performed using a range of enzyme concentrations. The heparanase enzyme reaction was allowed to proceed for various times prior to adding SA-XL665 and the emissions detected.
An HTRF Heparanase assay standard curve was performed using the conditions outlined in the product insert (figure 2A). Assay CVs were lower than 3%. An assay using 90 ng/ml heparanase was then run in the presence of suramin, a known inhibitor of heparanases. The inhibition curve is shown in figure 2B. The calculated IC50 was 4µM, which is in agreement with values calculated using radiometric methods (1-10µM).
HTRF Heparanase assay was performed using a range of enzyme concentrations following standard assay conditions with a final assay volume of 20 µl. (B) The heparanase assay was performed using 90ng/ml heparanase and incubated for 30 minutes at room temperature in the presence of various concentrations of Suramin.
Nuclear receptor ligand identification with HTRF find_in_pageCisbio | Download |
HTRF assays handle low- to high affinity protein-protein interactions find_in_pageCisbio | Download |
Protein-Protein Interaction Assays with HTRF find_in_pageCisbio Bioassays, Codolet, France . | Download |
Guidelines for optimizing protein-protein interaction assays using HTRF PPI reagents find_in_pageCisbio, Codolet, France | Download |
Truly homogeneous functional assays
FRET (Fluorescence Resonance Energy Transfer) is based on the transfer of energy between two fluorophores – a donor and an acceptor – when in close proximity. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and by detecting the level of energy transfer.
