Recombinant Human Hyaluronidase 4/HYAL4 Protein, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
Cys34-Pro462, 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.
- Dilute rmCHST3 to 0.3 mg/mL in Labeling Buffer.
- Combine 200 µL Labeling Buffer, 60 µL diH2O, 60 µL PAP35S, 40 µL Chondroitin Sulfate, and 40 µL rmCHST3 and incubate mixture at 37 °C for 2 hours.
- Add 200 µL diH2O to incubated rxn mixture for a final volume of 600 µL (rxn mix sufficient for ~40 rxns).
- Dilute rhHYAL4 to 666.7, 200, 33.33, 13.33, 6.667, 2.0, 0.333, and 0.00667 µg/mL in Assay Buffer.
- Combine 15 µL of rhHYAL4 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 hr. 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. Mark a second line just below the loading dye migration front.
- Using the developed film as an overlay, mark a third line where the labeled product migrated (ignore any free sulfate which will appear equally in all lanes and will have migrated the furthest). Hint: 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 top of the gel and the dye front is considered to contain the labeled starting material. The area between the dye front and the product line 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.
- Add 5 mL liquid scintillation fluid to each vial and count the vials for 35S.
- Determine the amount of rhHYAL4 required for 50% cleavage by plotting % cleavage (control adjusted) vs. ng of rhHYAL4 with 4‑PL fitting.
- Chondoitin Sulfate: 10 ug
- rhHYAL4: 10000, 3000, 500, 200, 100, 30, 5, and 0.1 ng
Background: Hyaluronidase 4/HYAL4
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 the only known endo-beta -N-acetyl-galactosaminidase that is mainly active on chondroitin sulfate (type C and D in particular) with no activity on hyaluronan (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 ‑3GalNAc beta 1‑ most closely resembles to that of hyaluronan composed of repeating units of ‑4GlcA beta ‑3GlcNAc beta 1‑, which may partially explain the overlapping substrate specificity of most hyaluronidases. HYAL4 is believed to specifically degrade chondroitin sulfate in the lysosome (8), as its optimal pH for activity is around 4.5 (6). Accordingly, the enzymatic activity was measured by treating 35S-labeled chondroitin sulfate with recombinant HYAL4 (9). HYAL4 may be used to investigate the biological roles of chondroitin sulfate in tissues and cells.
- Csoka, A.B. et al. (2001) Matrix Biol. 20:499.
- Jedrzejas, M. J. and Stern, R. Proteins (2005) 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.
- Prabhakar, v. and Sasisekharan, R. (2006) Adv. Pharmacol. 53:69.
- Ethen, C.M. et al. http://www.rndsystems.com/resources/images/21388.pdf
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Glycan Detection Reagents
Sulfotransferase Assays and Substrates
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