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Native PAGE eliminates the problem of PEG–SDS interaction in SDS‐PAGE and provides an alternative to HPLC in characterization of protein PEGylation
Author(s) -
Zheng Chunyang Y.,
Ma Guanghui,
Su Zhiguo
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600807
Subject(s) - pegylation , peg ratio , chromatography , high performance liquid chromatography , chemistry , polyacrylamide gel electrophoresis , characterization (materials science) , size exclusion chromatography , resolution (logic) , polyethylene glycol , biochemistry , materials science , nanotechnology , enzyme , computer science , finance , artificial intelligence , economics
PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP‐HPLC fails to give the correct information about the reaction of PEG 20000. Size‐exclusion HPLC (SE‐HPLC) produced very poor resolution on the PEG 5000 reaction. SDS‐PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG–SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS‐PAGE for the analysis of PEG–protein conjugates especially for characterization of the PEGylation mixture.