Recombinant Human Hyaluronidase 2/HYAL2 His-tag Protein, CF
Recombinant Human Hyaluronidase 2/HYAL2 His-tag Protein, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
The majority of Cy5-Labeled Hyaluronan (Low MW) (2 pmol) is digested by 0.25 μg of Recombinant Human HYAL-2, as measured under the described conditions.
Met21-Gly447, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
11012-GH
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM MES, pH 5.0
- Recombinant Human HYAL-2 (rhHYAL-2) (Catalog # 11012-GH)
- Cy5-Labeled Hyaluronan (Low MW) (Cy5 LMW-HA) (Catalog # GL401)
- 17% SDS-PAGE gel and SDS-PAGE Reagents
- Reducing SDS-PAGE sample buffer
- Fluorescent imager
- Dilute rhHYAL-2 to 25 ng/µL in Assay Buffer.
- Dilute Cy5 LMW-HA to 0.2 µM in Assay Buffer.
- Prepare reaction by combining 10 µL of diluted rhHYAL-2 and 10 µL of diluted Cy5 LMW-HA.
- Prepare a negative control by combining 10 µL of diluted Cy5 LMW-HA and 10 µL of Assay Buffer.
- Incubate reaction(s) and negative control at room temperature for 18 hours.
- Add 7 µL of the reducing SDS-PAGE sample buffer to each reaction and negative control.
- Load 13.5 µL each sample and control per well on a 17% SDS-PAGE gel and run SDS-PAGE until the dye front has migrated more than two thirds of the way down the gel.
- Acquire gel image with a fluorescent imager.
- Visually determine the digestion of Cy5 LMW-HA by rhHYAL-2 to dp 1-4 in each reaction lane.
- rhHYAL-2: 0.25 µg
- Cy5 LMW-HA: 2 pmol
Scientific Data
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rhHYAL-2 recognizes repeating subunits of -GlcA-beta 1,3-GlcNAc-beta 1,4- and hydrolyzes high molecular weight hyaluronic acid to intermediate and smaller sized products.
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1 μg/lane of rhHYAL-2 (Catalog # 11012-GH) was resolved with SDS-PAGE under reducing (R) conditions and visualized by Silver Staining, showing a band at 55 kDa.
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Lane 1 contained substrate Cy5-labeled LMW-HA GL401. In the presence of rhHYAL-2, the Hyaluronic Acid was modified to smaller molecular weight products with increasing amounts of rhHYAL-2 from left to right and a mobility shift was observed.
Reconstitution Calculator
Background: Hyaluronidase 2/HYAL2
Human hyaluronidases (HYALs) are a group of five endo-beta -N-acetylhexosaminidases that include HYAL1, HYAL2, HYAL3, HYAL4, and SPAM1 (PH20) (1, 2). While HYAL1, HYAL2 and SPAM1 are mainly active on hyaluronan (3, 4, 5), HYAL4 is active on both hyaluronan (6) and chondroitin sulfate (type C and D in particular) (7). Hyaluronan and chondroitin sulfate are abundant extracellular matrix components that have numerous biological functions (8). The backbone of chondroitin sulfate composed of repeating units of 4-GlcA-beta 1,3-GalNAc-beta 1 most closely resembles to that of hyaluronan composed of repeating units of 4-GlcA-beta 1,3-GlcNAc-beta 1. While HYAL1 is a strict lysosomal hyaluronidase and is active when pH ≤ 5.5 (9), HYAL2 is active in a narrow range of pH 5.0 to 5.5 (6) and is attached to cell membrane via GPI linker (10) where they may serve to hydrolyze high molecular weight hyaluronic acid of extracellular matrix to intermediate and small sized products.
- Csoka, A.B. et al. (2001) Matrix Biol. 20:499.
- Jedrzejas, M. J. and Stern, R. (2005) Proteins 61:227.
- Csoka, A.B. et al. (1999) Genomics 60:351.
- Lin, Y. et al. (1994) J. Cell Biol.125:1157.
- Lepperdinger, G. et al. (2001) Matrix Biol. 20:509.
- Wu, Z.L. and Ertelt, J.M. (2021) Glycobiology, cwab061, https://doi.org/10.1093/glycob/cwab061.
- Kaneiwa, T. et al. (2010) Glycobiology 20:300.
- Iozzo, R.V. (1998) Annu. Rev. Biochem. 67:609.
- Hofinger, E.S.A. et al. (2007) Glycobiology 17:444.
- Rai, S.K. et al. (2001). Proc. Natl. Acad. Sci. U.S.A, 98:4443.
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