Cy5 Labeled Hyaluronan (Low MW)

Catalog # Availability Size / Price Qty
GL401-200
Cy5 Labeled Hyaluronan (Low MW).
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Cy5 Labeled Hyaluronan (Low MW) Summary

GDP-Cy3-Fucose for fucose detection

 

Formulation

Supplied in 20 mM Tris, pH 8.0

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

Enzymes and detection reagents

 

Data Examples

Recombinant HYAL1 assay using Cy5 Labeled Hyaluronan (Low MW)/(Cy5-LMW HA) as a substrate
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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

Source
Synthetic
Shipping Conditions
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Product Datasheets

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Scientific Data

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

  1. Dilute rhHYAL1 to 1 ng/µL in Assay Buffer.
  2. Complete eight 2-fold serial dilution using 10 µL of the diluted rhHYAL1 (10 ng). This has a range of 10-0.08 ng.
  3. Dilute Cy5-LMW HA to 0.2 µM in Assay Buffer.
  4. Add 10 µL of the Cy5-LMW HA to each dilution.
  5. Prepare a negative control by mixing 10 µL of the Cy5-LMW HA with 10 µL of Assay Buffer.
  6. Incubate the reaction and control at 37°C for 20 minutes.
  7. Stop the reactions and controls by adding 5 µL of 6X gel loading dye to each of the above tubes.
  8. 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.
  9. 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

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