Fluorescent labeling of proteins and its application to SDS-PAGE and western blotting.
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[alpha]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 later, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.
Department de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193, Bellaterra (Barcelona), Spain.
F Javier
FJ
Alba
Salvador
S
Bartolomé
Antonio
A
Bermúdez
Joan-Ramon
JR
Daban
D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010078:Oxazines / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D005465:Fluorocarbon Polymers
D005456:Fluorescent Dyes; D010078:Oxazines; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C044808:nile red