Cy5 Labeled Heparan Sulfate Proteoglycan (Cy5-HSPG)
R&D Systems, part of Bio-Techne | Catalog # GL402
Key Product Details
Summary for Cy5 Labeled Heparan Sulfate Proteoglycan (Cy5-HSPG)
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| Formulation | Supplied in 25 mM Tris, 150 mM NaCl, pH 7.5 |
| Stability & Storage | Store at |
Applications
- Used as a substrate to study activity of various heparanases and heparinases.
- Used to study protein heparan sulfate interactions.
Key Features:
- Excitation at 649 nm and emission at 671 nm.
- The fluorescent dye Cy5 is conjugated to the non-reducing end terminal GlcNAc residue.
- Heparan sulfate can be released by digestion with heparanase or heparinase II, heparinase III.
- Can be separated on 15%-17% SDS-PAGE and directly visualized.
For Details:
Related Reagents
Click Chemistry
- UDP-Azido-GlcNAc (ES104)
- GDP-Cy5-Fucose (ES301)
- CMP-Cy5-Sialic Acid (ES302)
- GDP-Cy3-Fucose (ES401)
- CMP-Cy3-Sialic Acid (ES402)
Enzymes and detection reagents
- Recombinant Human Active Heparanase/HPSE Protein (7570-GH)
- Recombinant Mouse Active Heparanase/HPSE Protein (9788-GH)
- Recombinant Human Exostosin 1/2 Heterodimer Protein (8567-GT)
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Cy5 Labeled Heparan Sulfate Proteoglycan (Cy5-HSPG). This substrate can be used to study the activity of various heparanases and heparinases. This can also be used to study protein heparan sulfate interactions. The fluorescent dye Cy5 is conjugated to the non-reducing end terminal GlcNAc residue. |
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Fluorescent gel mobility shift caused by Recombinant Mouse Heparanase. Lane 1-4 contained Cy5 Labeled Heparan Sulfate Proteoglycan (Cy5-HSPG) (Catalog # GL402) digested with increasing amounts of Recombinant Mouse Active Heparanase/HSPE Protein (9788-GH). Lane 5 contained Cy5-HSPG only. Lane 6, Cy5-HSPG was digested with Recombinant P. heparinus Heparinase III Protein (6145-GH). Lane 7, Cy5-HSPG was digested with Recombinant P. heparinus Heparinase II Protein (6336-GH). |
Assay Procedure
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
- Dilute rmHPSE to 100 ng/µL and Cy5-HSPG to 0.4 µM with Assay Buffer.
- Add 5 µL of rmHPSE, 5 µL of Cy5-HSPG, and 10 µL of Assay Buffer to a tube.
- Prepare a negative control by mixing 5 µL of Cy5-HSPG and 15 µL of Assay Buffer in another tube.
- Incubate the reaction and control at 37o C for 60 minutes.
- Add 5 µL of 6X Gel Loading Dye to each tube.
- 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.
- Expose the gel for 10 seconds on a fluorescent imager.
- Determine change in fluorescent signal with Alphaview or any other densitometry software.
Final Assay Conditions Per Reaction
- rmHPSE: 500 ng
- Cy5-HSPG: 2 pmol
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