A Simple and Rapid Immunoassay System Using Green Fluorescent Protein Tag

A fusion protein between green fluorescent protein (GFP) and neuron-specific enolase (NSE) was expressed in Escherichia coli. The GFP-NSE fusion protein migrated at 62 kDa in SDS-PAGE and retained the fluorescence under non-heating conditions. However, heat-denatured GFP-NSE was non-fluorescent and...

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Bibliographic Details
Published in:Journal of immunoassay (Monticello, N.Y.) N.Y.), 1997-11, Vol.18 (4), p.321-333
Main Authors: Aoki, Takashi, Kaneta, Mitsuhiro, Onagi, Hitoshi, Takahashi, Yasumitsu, Koch, Katherine S., Leffert, Hyam L., Watabe, Hiroyuki
Format: Article
Language:English
Subjects:
Animals
Escherichia coli
fluorescence immunoassay
Fluorescent Antibody Technique, Indirect
fusion protein
GFP-tagging
green fluorescent protein
Green Fluorescent Proteins
Humans
Indicators and Reagents
Luminescent Proteins
Molecular Weight
neuron-specific enolase
Phosphopyruvate Hydratase - analysis
Rabbits
Recombinant Fusion Proteins - analysis
Sheep
Spectrometry, Fluorescence
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Summary:A fusion protein between green fluorescent protein (GFP) and neuron-specific enolase (NSE) was expressed in Escherichia coli. The GFP-NSE fusion protein migrated at 62 kDa in SDS-PAGE and retained the fluorescence under non-heating conditions. However, heat-denatured GFP-NSE was non-fluorescent and migrated at 74 kDa corresponding to the theoretical value. This suggests that the special structure of GFP, which is not denatured by SDS, influences its mobility in SDS-PAGE under non-heating conditions. The fluorescence intensity of GFP-NSE was measurable over a wide range by spectrophotometry or densitometry. The competitive immunoassay for NSE was performed using GFP-NSE as labeled antigen. Under our assay conditions, the working range of this system was about 2 - 60 ng. This simple and rapid fluorescence immunoassay (FIA) using GFP-tagged antigen may be applicable to many protein markers.
ISSN:0197-1522
DOI:10.1080/01971529708005825