Construction of a fusion protein between protein A and green fluorescent protein and its application to Western blotting

FEBS Letters

Leffert, Hyam L.¹  Koch, Katherine S.¹  Watabe, Hiroyuki²  Takahashi, Yasumitsu²  Aoki, Takashi² 

[1]Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA 92036-0636, USA

[2]Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-02, Japan

关键词: Green fluorescent protein;    Protein A;    Fusion protein;    Western blotting;    Dot blotting;    GFP;    green fluorescent protein;    PA-GFP;    fusion protein between protein A and green fluorescent protein;    CBB;    Coomassie brilliant blue;    POD;    peroxidase;    DAB;    3;    3′-diaminobenzidine;    R-NSE;    recombinant human neuron-specific enolase;   

DOI  :  10.1016/0014-5793(96)00289-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE. However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA-GFP, was used in Western and dot blots. The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening.

【 授权许可】

Unknown