Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines.
Protein detection on SDS gels or on 2-D gels must combine several features, such as sensitivity, homogeneity from one protein to another, speed, low cost, and user-friendliness. For some applications, it is also interesting to have a nonfixing stain, so that proteins can be mobilized from the gel for further use (electroelution, blotting). We show here that coelectrophoretic staining by fluorophores of the oxacarbocyanine family, and especially diheptyloxacarbocyanine, offers several positive features. The sensitivity is intermediate between the one of colloidal CBB and the one of fluorescent ruthenium complexes. Detection is achieved within 1 h after the end of the electrophoretic process and does not use any fixing or toxic agent. The fluorescent SDS-carbocyanine-protein complexes can be detected either with a laser scanner with an excitation wavelength of 488 nm or with a UV table operating at 302 nm. Excellent sequence coverage in subsequent MS analysis of proteolytic peptides is also achieved with this detection method.
Luche
Sylvie
S
CEA--Laboratoire de Biochimie et Biophysique des systèmes Intégrés, iRTSV/BBSI, CEA-Grenoble, Grenoble, France.
Lelong
Cécile
C
Diemer
Hélène
H
Van Dorsselaer
Alain
A
Rabilloud
Thierry
T
D002232:Carbocyanines / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D007525:Isoelectric Focusing; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002232:Carbocyanines; D005456:Fluorescent Dyes; D011506:Proteins