title: Polyacrylamide gel with switchable trypsin activity for analysis of proteins.
abstract: 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.
authors.forename: Fangjie
authors.initials: F
authors.lastname: Liu
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Chunli
authors.initials: C
authors.lastname: Wang
authors.forename: Zhengyan
authors.initials: Z
authors.lastname: Hu
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hongqiang
authors.initials: H
authors.lastname: Qin
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D011092:Polyethylene Glycols / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D059748:Proteolysis; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols; D014357:Trypsin