Anal Chem. 2013 Aug 6;85(15):7024-8. doi: 10.1021/ac4017693. Epub 2013 Jul 19.
Trypsin was immobilized on a variety of materials to improve digestion efficiency. However, because the immobilized trypsin will digest proteins during electrophoresis, direct immobilization of active trypsin in polyacrylamide gel will compromise the protein separation. To overcome this problem, here we report a novel polyacrylamide gel with switchable trypsin activity. It was prepared by copolymerization of the PEG-trypsin-aprotinin complex during the gel-casting step. Because the inhibitor aprotinin binds strongly with trypsin at alkaline pH, this novel gel does not display hydrolytic activity during electrophoresis. After electrophoresis, the activity of trypsin embedded in gel could be recovered by simply washing away the bound inhibitor at a low pH. It was demonstrated that this unique switchable activity design allowed high resolution of the complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis.
胰蛋白酶被固定在各种材料上以提高消化效率。然而，由于固定化的胰蛋白酶在电泳过程中会消化蛋白质，因此直接将活性胰蛋白酶固定在聚丙烯酰胺凝胶中会影响蛋白质的分离。为了解决这个问题，我们在这里报道了一种新型的具有可切换胰蛋白酶活性的聚丙烯酰胺凝胶。它是通过在凝胶浇铸步骤中共聚 PEG-trypsin-aprotinin 复合物制备的。由于抑制剂 aprotinin 在碱性 pH 值下与胰蛋白酶结合紧密，因此这种新型凝胶在电泳过程中不会显示水解活性。电泳后，通过简单地在低 pH 值下洗掉结合的抑制剂，就可以恢复嵌入凝胶中的胰蛋白酶的活性。结果表明，这种独特的可切换活性设计允许在电泳过程中对复杂的蛋白质混合物进行高分辨率分离，并在电泳后原位对分离的蛋白质进行高效消化。
