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Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate‐polyacrylamide‐based gels by using hydrophobic tail‐mediated enhancement of fluorescein luminescence
Author(s) -
Kang Chulhun,
Kim Hyun Jung,
Kang Donghoon,
Jung Duk Young,
Suh Myungkoo
Publication year - 2003
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.200305605
Subject(s) - fluorescein , sodium dodecyl sulfate , chemistry , staining , fluorescence , gel electrophoresis , polyacrylamide , polyacrylamide gel electrophoresis , luminescence , chromatography , silver stain , fluorescein isothiocyanate , electrophoresis , biophysics , biochemistry , microbiology and biotechnology , materials science , biology , polymer chemistry , physics , optoelectronics , quantum mechanics , genetics , enzyme
Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5‐dodecanoyl amino fluorescein, 5‐hexadecanoyl amino fluorescein, and 5‐octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein‐to‐protein variation than silver staining. This new staining method was also successfully applied to two‐dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining.