Cy5 Labeled Hyaluronan (Low MW) Summary
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Formulation |
Supplied in 20 mM Tris, pH 8.0 |
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Stability & Storage |
Store at < -20 °C. Good for 6 months from date of receipt. |
Applications
- Studying hyaluronan protein binding.
- Used as a substrate for various hyaluronan specific hydrolases and lyases.
Key Features and Benefits:
- Excitation at 649 nm and emission at 671 nm.
- The fluorescent dye Cy5 is conjugated to the non-reducing end GlcNAc residue through enzymatic conjugation.
- Can be separated on 15%-17% SDS-PAGE and directly visualized as a ladder containing 5-100 repeating units (RU) of the HA disaccharide GlcA-β,3-GlcNAc.
- Linear response range for Cy5 labeled glycans can be from 10 fmol to 100 pmol, depending on the sensitivity of detection.
- Store at < -20 °C. Good for 6 months from date of receipt.
For Details:
Wu et al., (2019) Glycobiology 29:750-754
Wu et al., (2020) Glycobiology 30:970-980
Related Reagents
Click Chemistry
- GDP-Cy5-Fucose (ES301)
- CMP-Cy5-Sialic Acid (ES302)
- GDP-Cy3-Fucose (ES401)
- CMP-Cy3-Sialic Acid (ES402)
Enzymes and detection reagents
- Various Hyaluronidases and Hyaluronan Lyase
- HA-binding Proteins
- Hyaluronan
Data Examples
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Recombinant HYAL1 assay using Cy5 Labeled Hyaluronan (Low MW)/(Cy5-LMW HA) as a substrate (Catalog # GL401) was digested with HYAL1 Recombinant Human Hyaluronidase 1/HYAL1 Protein (Catalog # 7358-GH), according to the above protocol and then separated on SDS-gel and visualized with a fluorescent imager. Without digestion, Cy5-LMW HA displays a range of bands from RU5 to about RU100. RU, repeating unit of the HA disaccharide GlcA-β,3-GlcNAc. At high dosage, rhHYAL1 digested all HA species to RU1-RU4 products. |
Specifications
Product Datasheets
Scientific Data
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Cy5 Labeled Hyaluronan (Low MW). This substrate can be used for various hyaluronan specific hydrolases and lyases. The fluorescent dye Cy5 is conjugated to the non-reducing end GlcNAc residue. (n, the number of repeating disaccharide units, ranges from about 5 to 100)
Assay Procedure
Sample Assay Protocol using Cy5 Labeled Hyaluronan (Low MW)/Cy5-LMW HA as substrate for Recombinant Human HYAL1 assay
Protocols are guidelines. Parameters need to be optimized by end users. Cy5-LMW HA contains multiple bands, and it is suggested to have 2 pmol for each HYAL1 digestion.
Materials
- Assay Buffer: 50 mM NaOAc, pH 4.0
- Recombinant Human Hyaluronidase 1/HYAL1 Protein (rhHYAL1) (Catalog # 7358-GH)
- Cy5 Labeled Hyaluronan (Low MW)/(Cy5-LMW HA) (GL401)
- 15 % SDS-PAGE
- 6X gel loading dye
- A fluorescent imager
Assay
- Dilute rhHYAL1 to 1 ng/µL in Assay Buffer.
- Complete eight 2-fold serial dilution using 10 µL of the diluted rhHYAL1 (10 ng). This has a range of 10-0.08 ng.
- Dilute Cy5-LMW HA to 0.2 µM in Assay Buffer.
- Add 10 µL of the Cy5-LMW HA to each dilution.
- Prepare a negative control by mixing 10 µL of the Cy5-LMW HA with 10 µL of Assay Buffer.
- Incubate the reaction and control at 37°C for 20 minutes.
- Stop the reactions and controls by adding 5 µL of 6X gel loading dye to each of the above tubes.
- Load 12.5 µL each of the above samples per well on an SDS-PAGE and run at 10 volts/cm until the dye front is more than two thirds of the gel.
- Visualize the gel with a fluorescent imager for 10 seconds.
Final Assay Conditions Per Reaction
- rhHYAL1: 10 to 0.08 ng
- Cy5-LMW HA: 2 pmol
FAQs
No product specific FAQs exist for this product, however you may
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