Efficient immobilization of proteins by modification of plate surface with polystyrene derivatives

Analytical Biochemistry
N SuzukiY C Lee

Abstract

Immobilization of proteins on microplate wells by simple adsorption (e.g., for ELISA) is convenient, but it can be inefficient, especially if proteins are hydrophilic or small in size. This problem was alleviated by the use of polyvinylbenzyl lactonoylamide (PVLA). PVLA is strongly adsorbed to the hydrophobic well surface, and its lactonamide part can be oxidized with periodate to generate aldehydo groups. Proteins are then immobilized covalently to the aldehydo groups by reductive amination under mild conditions. Using this method, henceforth termed the PVLA method, alkaline phosphatase (AP) was immobilized to microplates six- to sevenfold greater than by simple adsorption (as measured by activity). Similarly, the activity of immobilized mannose-binding protein A (MBP-A) was 4- to 8-fold higher by the PVLA method than by simple adsorption. The PVLA-coated plates needed as little as 200 ng of MBP-A per well to have a sufficient amount of MBP-A immobilized for the measurement of binding of 125I-labeled mannosylated bovine serum albumin (125I-Man-BSA), but unmodified plates required as much as 20 micrograms/well MBP-A to obtain the same response. Recommended conditions for the PVLA method are 40 microliters of 2 mg/ml of PVLA for...Continue Reading

Citations

Nov 20, 2015·The Analyst·Satish Balasaheb NimseTaisun Kim
Mar 15, 2006·Analytical and Bioanalytical Chemistry·Haiqiu LiZhongmin Su
Aug 16, 2011·Analytical and Bioanalytical Chemistry·Yaodong ZhangTingting Hei

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