A proprietary heparan sulfate substrate labeled with both biotin and Eu3+ Cryptate to monitor heparanase activity.
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A proprietary heparan sulfate substrate labeled with both biotin and Eu3+ Cryptate to monitor heparanase activity.
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In stock

Overview

HTRF heparan sulfate substrate labeled with both biotin and Eu3+ Cryptate used with HTRF SA-XL665, allows to monitor heparanase activity with the robustness of HTRF technology.

Heparanase is an enzyme that cleaves heparan sulfate, and thereby releases growth factors that are implicated in tumor cell proliferation, metastasis and angiogenesis.

Assay principle

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.

Assay protocol

The assay is run as a two-step process: 1.Heparanase enzymatic reaction: Mix together, the substrate, the heparanase and/or compounds, incubate for 30 min at 37°C (We recommend a time course study to determine the optimal stimulation time) 2.Heparanase activity detection: Add Streptavidin-XL665

Heparanase assay: enzymatic kinetic

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. 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.

Heparanase assay: inhibitory effect of suramin

Heparanase assay was performed using a range of enzyme concentrations with a final assay volume of 20 µL. Then an inhition assay was performed using heparanase at 90ng/mL incubated for 30 minutes at RT in presence of various concentrations of Suramin.

Fig A shows the Heparanase assay standard curve, assay CVs were lower than 3%. Fig.B shows the inhibitory effect of Suramin, a known inhibitor of heparanases. The IC50 was calculated at 4 µM, which is in agreement with values calculated using radiometric methods (1-10µM).

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