Enzyme assay of hyaluronan synthase

 Wash cultured cells three times with PBS (−) and harvest them by scraping.

 Centrifuge at 1,200 rpm for 5 min at 4˚C.

 Suspend the cell pellets in 1 mL of ice-chilled lysis buffer.

 Disrupt the cells by sonication for 1 min with a sonicator.

 Ultracentrifuge the cell lysate at 105,000 × g at 4˚C for 1 h to give high-speed pellets.

 Suspend the high-speed pellets with 0.2 mL of HAS reaction buffer.

 Incubate at 37˚C for 1 h.

 Further incubate aliquot (0.1 mL) of the sample with 1 TRU of Streptomyces hyaluronidase.

 Stop the reaction by addition of SDS to 2% (w/v).

 Spot the mixtures onto Whatman no. 3MM paper.

 Transfer the paper to a paper chromatography chamber.

 Perform descending paper chromatography in the solvent containing 1 M ammonium acetate (pH 5.5) and ethanol (65:35 v/v) for three days.

 Cut the origin containing the synthesized polymers from the paper.

 Determine the amount of radioactivity in the high molecular mass hyaluronan by liquid scintillation counting.

 Incubate the synthesized hyaluronan at 37˚C for 1 h with or without 1 TRU of Streptomyces hyaluronidase.

 Mix 14 μL of the hyaluronan sample with 2 μL of 2 M sucrose/TAE buffer.

 Prepare a 0.5% (w/v) agarose gel (20 × 20 cm) by melting 0.6 g agarose in 108 mL of TAE buffer.

 Transfer the gel plate to the horizontal electrophoresis apparatus.

 Apply the sample to each lane of the agarose gel.

 Electrophorese at 2V/cm for 10 h in TAE buffer at room temperature.

 Stain the gel for 4 h under light-protective cover at room temperature in a staining solution.

 Destain the gel in water and then dry.

 Photograph and detect the radioactive HA on x-ray film.