title: Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.
abstract: This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described below, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.
authors.affiliation: Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 08193, Spain.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Alba
authors.forename: Salvador
authors.initials: S
authors.lastname: Bartolomé
authors.forename: Antonio
authors.initials: A
authors.lastname: Bermúdez
authors.forename: Joan-Ramon
authors.initials: JR
authors.lastname: Daban
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005663:Furans / Q000737:chemistry; D006801:Humans; D010078:Oxazines / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D005663:Furans; D010078:Oxazines; D011506:Proteins; C008394:2-methoxy-2,4-diphenyl-3(2H)-furanone; C044808:nile red