Cy5 Labeled Hyaluronan (Low MW)
R&D Systems, part of Bio-Techne | Catalog # GL401
Key Product Details
Summary for Cy5 Labeled Hyaluronan (Low MW)
|
| |
| Formulation | Supplied in 20 mM Tris, pH 8.0 |
| Stability & Storage | Store at |
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
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
|
| 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. |
Hyaluronan, also known as hyaluronic acid (HA) or sodium hyaluronate, is a naturally occurring linear polymer. Hyaluronan is a glycosaminoglycan (GAG) that is ubiquitously present in the extracellular matrix of all vertebrates and is also present in the capsule of some strains of Streptococci. Mammalian hyaluronan is synthesized by one of three distinct hyaluronan synthases (HAS1, 2, and 3), which produce HA polymers with different chain lengths and differ in their rates of synthesis. Whereas high molecular weight HA (> 500 kDa) is anti-angiogenic, anti-inflammatory and immunosuppressive, low molecular weight HA (10 - 500 kDa) is highly angiogenic and pro-inflammatory. HA oligomers are anti-apoptotic and upregulate heat shock protein expression. Functionally, hyaluronan molecules are important for the maintenance of a highly hydrated extracellular matrix in tissues, which is involved in cell adhesion and supports cell migration.
|
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
Customer Reviews for Cy5 Labeled Hyaluronan (Low MW)
There are currently no reviews for this product. Be the first to review Cy5 Labeled Hyaluronan (Low MW) and earn rewards!
Have you used Cy5 Labeled Hyaluronan (Low MW)?
Submit a review and receive an Amazon gift card!
$25/€18/£15/$25CAN/¥2500 Yen for a review with an image
$10/€7/£6/$10CAN/¥1110 Yen for a review without an image
Submit a review