Cy5 Labeled Heparan Sulfate Proteoglycan (Cy5-HSPG)

Sample Assay Protocol for using Cy5 Labeled Heparan Sulfate Proteoglycan (Cy5-HSPG) as substrate for Recombinant Mouse Heparanase Assay

Protocols are guidelines. Parameters need to be optimized by end users. Suggested input of Cy5-HSPG in an assay separated on SDS-PAGE is 2 pmol.

Materials

• Assay Buffer: 50 mM NaOAc, pH 5.0
• Recombinant Mouse Active Heparanase/HPSE Protein (rmHPSE) (Catalog # 9788-GH)
• Cy5 Labeled Heparan Sulfate Proteoglycan (Cy5-HSPG) (R&D Systems®, Catalog # GL402)
• 15% SDS-PAGE
• 6X Gel Loading Dye
• A fluorescent imager

Assay

1. Dilute rmHPSE to 100 ng/µL and Cy5-HSPG to 0.4 µM with Assay Buffer.
2. Add 5 µL of rmHPSE, 5 µL of Cy5-HSPG, and 10 µL of Assay Buffer to a tube.
3. Prepare a negative control by mixing 5 µL of Cy5-HSPG and 15 µL of Assay Buffer in another tube.
4. Incubate the reaction and control at 37^o C for 60 minutes.
5. Add 5 µL of 6X Gel Loading Dye to each tube.
6. Load 12.5 µL of each tube to a 15% SDS-PAGE gel and run the gel at 250 constant voltage until the dye front reaches the end of the gel.
7. Expose the gel for 10 seconds on a fluorescent imager.
8. Determine change in fluorescent signal with Alphaview or any other densitometry software.

Final Assay Conditions Per Reaction

• rmHPSE: 500 ng 
• Cy5-HSPG: 2 pmol