Fluorescent labeling of proteins with nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots.
The fluorescent hydrophobic dye Nile red allows the rapid, sensitive, and general staining of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Nile red staining does not preclude further electroblotting of protein bands onto polyvinylidene difluoride (PVDF) membranes. The resulting Western blot can be stained with the covalent fluorescent dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) using a simple procedure. MDPF staining allows further N-terminal microsequencing and immunodetection of specific bands. This review considers the physicochemical, structural, and analytical studies that have led to the development of Nile red and MDPF staining methods. The usefulness of these procedures is discussed in comparison to other currently available fluorescent and nonfluorescent protein detection methods.
Daban
J R
JR
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, Ballaterra, Spain. joanramon.daban@uab.es
D015153:Blotting, Western; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005663:Furans / Q000737:chemistry; D010078:Oxazines / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D020539:Sequence Analysis, Protein; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods
D005456:Fluorescent Dyes; D005663:Furans; D010078:Oxazines; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; C008394:2-methoxy-2,4-diphenyl-3(2H)-furanone; C044808:nile red