Premium
Recombinant autofluorescent landmarks for standardization of electrophoretic migration of proteins
Author(s) -
Einhauer Adelheid,
Jungbauer Alois
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200204)23:7/8<1146::aid-elps1146>3.0.co;2-i
Subject(s) - isoelectric focusing , electrophoresis , isoelectric point , gel electrophoresis , proteome , two dimensional gel electrophoresis , fusion protein , chromatography , protein purification , protein tag , heat shock protein , chemistry , recombinant dna , biochemistry , biology , proteomics , microbiology and biotechnology , enzyme , gene
Abstract Unequivocal identification of unknown protein spot patterns in two‐dimensional (2‐D) electrophoresis still represents a major problem when performing comparative studies of different 2‐D electrophoresis gels. Inhomogeneity of gels due to variations in the gel casting procedure, electroendoosmosis and heterogeneity of proteins are major contributions to variations in migration patterns. By fusing green fluorescent protein to a number of well‐defined selected proteins (human lysozyme, initiation factor 5a (EIF5a), rapamycin‐selective 25 kDa immunophilin (FKBP25), and heat shock protein 90 beta (hsp90)), the isoelectric points and the molecular mass were designed. Proteins were additionally tagged with the FLAG tag enabling rapid purification by immunoaffinity chromatography. The fusion proteins were expressed intracellularly in yeast to avoid heterogeneity caused by post‐translational modifications. The quality and applicability was tested in 1‐D and 2‐D electrophoresis. Sharp bands or symmetric spots were obtained. The proteins are considered as a new generation of reference proteins for electrokinetic separation methods.