The activity of proteases may be lost during microquantification because of their nonspecific binding to tubes and pipette tips. In this study, the effects of blocking reagents (blockers) in dilutions of digestive proteases of yellowtail, Seriola quinqueradiata , were compared. EzBlock Chemi (EBC; protein free), Western BLoT Blocking Buffer (WBB; protein free), casein-based blocker, and bovine serum albumin were evaluated by the measurement of trypsin, chymotrypsin, and aminopeptidase activities using fluorogenic substrates. Protease activities were linear in extracts of pyloric caeca at dilution rates of up to at least 1/6400 with the four blockers. However, the activities of pancreatic enzymes differed, and the highest levels were observed with EBC and WBB. Extracts diluted with EBC and WBB were incubated for up to 24 h at 25 °C. Pancreatic enzyme activities increased with time in extracts diluted with EBC, but for those diluted with WBB there was no clear trend. The activities of chymotrypsin and aminopeptidase in whole-body extracts of individual yellowtail larvae extracted with EBC were significantly higher than in those extracted with 150 mM NaCl, suggesting that EBC inhibits the loss of enzyme activity that may otherwise occur during extraction. Thus, EBC is considered to be the most effective diluent for the microquantification of proteases amongst the four blockers tested.

中文翻译：

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用不含蛋白质的封闭剂稀释消化鱼蛋白酶可防止微量定量过程中催化活性的损失

由于蛋白酶与管和移液器吸头的非特异性结合，在微量定量过程中可能会丧失蛋白酶的活性。在这项研究中，比较了封闭剂（封闭剂）在黄尾鱼消化蛋白酶稀释液中的作用。EzBlock Chemi（EBC；无蛋白质）、Western BLoT 封闭缓冲液（WBB；无蛋白质）、基于酪蛋白的阻断剂和牛血清白蛋白通过使用荧光底物测量胰蛋白酶、胰凝乳蛋白酶和氨肽酶活性进行评估。蛋白酶活性在幽门盲肠提取物中呈线性，使用四种阻滞剂稀释率高达至少 1/6400。然而，胰酶的活性不同，EBC 和 WBB 的水平最高。用 EBC 和 WBB 稀释的提取物在 25°C 下孵育长达 24 小时。在用 EBC 稀释的提取物中，胰酶活性随时间增加，但对于用 WBB 稀释的提取物，没有明显的趋势。用 EBC 提取的个体黄尾鱼幼虫全身提取物中糜蛋白酶和氨肽酶的活性显着高于用 150 mM NaCl 提取的那些，表明 EBC 抑制了酶活性的丧失，否则可能会在提取过程中发生。因此，在测试的四种阻断剂中，EBC 被认为是蛋白酶微量定量最有效的稀释剂。用 EBC 提取的个体黄尾鱼幼虫全身提取物中糜蛋白酶和氨肽酶的活性显着高于用 150 mM NaCl 提取的那些，表明 EBC 抑制了酶活性的丧失，否则可能会在提取过程中发生。因此，在测试的四种阻断剂中，EBC 被认为是蛋白酶微量定量最有效的稀释剂。用 EBC 提取的个体黄尾鱼幼虫全身提取物中糜蛋白酶和氨肽酶的活性显着高于用 150 mM NaCl 提取的那些，表明 EBC 抑制了酶活性的丧失，否则可能会在提取过程中发生。因此，在测试的四种阻断剂中，EBC 被认为是蛋白酶微量定量最有效的稀释剂。