Recombinant Human Hyaluronidase 1/HYAL1 Protein, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
Met1-Trp435, with a C-terminal 6-His tag
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|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.
- Labeling Buffer: 25 mM MES, 0.5% (v/v) Triton® X-100, 2.5 mM MgCl2, 2.5 mM MnCl2, 1.25 mM CaCl2, 0.75 mg/mL BSA, pH 7.0
- Assay Buffer: 0.1 M NaOAc, pH 4.5
- Gel Running Buffer: 40 mM Tris, 1 mM EDTA, adjust to pH 8.0 with acetic acid
- Recombinant Human Hyaluronidase 1/HYAL1 (rhHYAL1) (Catalog # 7358-GH)
- Recombinant Mouse Carbohydrate Sulfotransferase 3/CHST3 (rmCHST3) (Catalog # 5356-ST)
- 8% SDS-PAGE (approximately 15 cm x 20 cm, 20 lanes per gel)
- Hyaluronan (Catalog # GLR004), 20 mg/mL
- PAP35S (prepared in-house using the PAPS Synthesis Kit (Catalog # EA005), ~1 μM and ~2 x 106 cpm/μL)
- Gel loading buffer: 0.15 M Tris, 20.8 mM SDS, 1.15 M Glycine, 174 µM Bromophenol Blue, 30% Glycerol
- Blotting paper (Fisher Sci., Catalog # 05-714-4)
- Gel dryer
- Glogos® II autorad markers (Stratagene, Cat. # 420202) or equiv.
- Blue sensitive medical X-ray film
- X-ray film cassette
- Film developer (Konica SRX-101A Medical Film Processor) or equiv.
- Liquid scintillation counter (Beckman Coulter, Model # LS5000TD) or equiv.
- Liquid scintillation fluid (Beckman Coulter, Catalog # 141349) or equiv.
- Dilute rmCHST3 to 0.3 mg/mL in Labeling Buffer and Hyaluronan to 10 mg/mL in diH2O.
- Combine 200 µL Labeling Buffer, 60 µL diH2O, 60 µL PAP35S, 40 µL Hyaluronan, and 40 µL rmCHST3 and incubate mixture at
37 °C for 4 hours.
- Add 200 µL diH2O to incubated rxn mixture for a final volume of 600 µL (rxn mix sufficient for ~ 40 rxns).
- Dilute rhHYAL1 to 15, 4.165, 2.08, 1.04, 0.52, 0.26, 0.13, and 0.00667 µg/mL in Assay Buffer.
- Combine 15 µL of rhHYAL1 at each dilution with 15 µL rxn mix. Include a control containing 15 µL Assay Buffer and 15 µL rxn mix. Incubate at 37 °C for 20 min.
- Add 15 µL gel loading buffer to each rxn. Mix.
- Load 30 µL of each rxn per lane on a gel. Leave empty lanes between samples. Run at 200 V for 35 min.
- Transfer gel onto blotting paper and dry with gel dryer for 1 hour or until fully dry.
- Affix two autorad markers to the blotting paper next to the dried gel.
- In a darkroom expose dried gel to X-ray film by enclosing overnight in a cassette. Develop the film the following day.
- Using the dried gel, begin marking regions to be cut out for scintillation counting. Mark a horizontal line across the top of the entire gel just under the bottom of the loading wells.
- Using the developed film as an overlay, mark a second line below the lower edge of the labeled hyaluronan.
- Draw a third line just below where the labeled product migrated (ignore any free sulfate, appearing equivalent in all lanes, and migrating the furthest). Note: It will be easiest to use the highest enzyme-containing lanes to identify the product. For the control, identify the empty region where the product would appear.
- The area between the first two lines is considered to contain the labeled starting material. The area between the second two lines is considered to contain the compact cleavage product resulting from the reaction.
- Mark vertical lines distinguishing one lane (reaction condition) from another.
- Cut each region (two per lane) and place each into a separate liquid scintillation vial.
- Determine the amount of rhHYAL1 required for 50% cleavage by plotting % cleavage vs. ng of rhHYA-L1 with 4-PL fitting.
- Hyaluronan: 10 μg
- rhHYAL1: 225, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95 and 0.1 ng
Background: Hyaluronidase 1/HYAL1
Human hyaluronidases (HYALs) are a group of five endo‑ beta ‑N‑acetyl‑hexosaminidases that include HYAL1, HYAL2, HYAL3, HYAL4, and SPAM1 (PH20) (1, 2). While HYAL1, HYAL2 and SPAM1 are endo‑ beta ‑N‑acetyl‑glucosaminidase that are mainly active on hyaluronan with little activity on chondroitin sulfate (3, 4, 5), HYAL4 is mainly active on chondroitin sulfate (type C and D in particular) (6). Hyaluronan and chondroitin sulfate are abundant extracellular matrix components that have numerous biological functions (7). The backbone of chondroitin sulfate composed of repeating units of ‑4GlcA beta 1‑3GalNAc beta 1‑ most closely resembles to that of hyaluronan composed of repeating units of ‑4GlcA beta 1‑3GlcNAc beta 1‑, which may partially explain the overlapping substrate specificity of most hyaluronidases. HYAL1 is a lysosomal hyaluronidase with optimal activity around pH 4.0 (8). It is highly expressed in the liver, kidney and heart, and is the predominant hyaluronidase found in plasma (9). Defects in HYAL1 are associated with mucopolysaccharidosis type IX, or hyaluronidase deficiency (10). Surprisingly, HYAL1 has been reported both as a tumor promoter and suppressor (11, 12).The enzymatic activity of recombinant human HYAL1 was measured using 35S‑labeled hyaluronan as substrate.
- 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.
- 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.
- Frost, GI. et al. (1997) Biochem. Biophys. Res. Commun. 236:10.
- Natowicz, M.R. et al. (1996) N. Engl. J. Med. 335:1029.
- Nykopp, T.K. et al. (2009) BMC Cancer 9:143.
- Lokeshwar, V.B. (2005) Cancer Res. 65:7782.
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Glycan Detection Reagents
Sulfotransferase Assays and Substrates
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