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Construction of a fusion protein between protein A and green fluorescent protein and its application to Western blotting
Author(s) -
Aoki Takashi,
Takahashi Yasumitsu,
Koch Katherine S.,
Leffert Hyam L.,
Watabe Hiroyuki
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00289-x
Subject(s) - green fluorescent protein , fusion protein , fluorescence , aequorea victoria , blot , escherichia coli , molecular mass , chemistry , microbiology and biotechnology , biochemistry , biology , biophysics , recombinant dna , gene , enzyme , physics , quantum mechanics
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.