Rapid fluorescent monitoring of total protein patterns on sodium dodecyl sulfate-polyacrylamide gels and western blots before immunodetection and sequencing.
The fluorogenic dye 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) has been used for the detection of total protein patterns on polyvinylidene difluoride (PVDF) membranes. Fluorescent staining of protein bands on membranes with this covalent dye is completed in 20 min. Wet membranes are translucent, allowing protein visualization by transillumination with ultraviolet light. The resulting images can be recorded using Polaroid film or a charge-coupled device camera. Electrophoretic bands containing 5-10 ng of protein can be detected on the MDPF-stained Western blot. When proteins are directly transferred to the membrane using a slot blotting device, as little as 0.5 ng of protein can be detected. Previous visualization of protein bands on sodium dodecyl sulfate-polyacrylamide gels with the noncovalent fluorescent dye Nile red (Alba et al., BioTechniques, 1996, 21, 625-626) does not interfere with further MDPF staining and fluorescent detection of these bands transferred to PVDF membranes. Thus, Nile red and MDPF staining can be performed sequentially, allowing the rapid monitoring of total protein patterns on both the electrophoretic gel and Western blot. Using the conditions described in this study, MDPF staining does not preclude further N-terminal microsequencing and immunodetection of specific bands with polyclonal antibodies.
Alba
F J
FJ
Department de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Daban
J R
JR
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005663:Furans; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis; D010047:Ovalbumin / Q000032:analysis; D010078:Oxazines; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D017421:Sequence Analysis; D012710:Serum Albumin, Bovine / Q000032:analysis; D014362:Trypsinogen / Q000032:analysis; D005465:Fluorocarbon Polymers
D005456:Fluorescent Dyes; D005663:Furans; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010047:Ovalbumin; D010078:Oxazines; D011145:Polyvinyls; D011506:Proteins; D012710:Serum Albumin, Bovine; D014362:Trypsinogen; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C008394:2-methoxy-2,4-diphenyl-3(2H)-furanone; C044808:nile red