Hyaluronidase activity of sTMEM2 (Filtration)

Ultrafiltration (molecular weight cut-off 50 kDa) of 0.1 μg/ml fluorescein-labelled high-molecular-weight hyaluronan (HMW-HA, ~800 kDa, Carbosynth) incubated overnight with 100 μg/ml transmembrane protein 2 ectodomain (sTMEM2) or sperm hyaluronidase (HAase, Sigma-Aldrich, H3884). The reaction buffer was 100 mM MES, pH 6.0, 1 mM CaCl2. The mock condition contained an irrelevant protein at 100 μg/ml (Endo180 domains 1-4).

将0.1 μg/mL的荧光素标记高分子量透明质酸（fluorescein-labelled high-molecular-weight hyaluronan, HMW-HA，分子量约800 kDa，购自Carbosynth）与100 μg/mL的跨膜蛋白2胞外域（transmembrane protein 2 ectodomain, sTMEM2）或精子透明质酸酶（sperm hyaluronidase, HAase，Sigma-Aldrich，货号H3884）在反应缓冲液（reaction buffer）中过夜孵育后，采用截留分子量（molecular weight cut-off）为50 kDa的超滤（Ultrafiltration）进行处理。所用反应缓冲液为100 mM MES，pH值为6.0，且含有1 mM氯化钙。阴性对照条件（mock condition）组中加入了100 μg/mL的无关蛋白Endo180结构域1-4。