Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines

Abstract

Protein detection on SDS gels or on two dimensional 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 non fixing stain, so that proteins can be mobilized from the gel for further use (electroelution, blotting). We show here that co-electrophoretic staining by fluorophores of the oxacarbocyanine family, and especially diheptyloxacarbocyanine, offers several positive features. The sensitivity is intermediate between the one of colloidal Coomassie blue and the one of fluroescent ruthenium complexes. Detection is achieved within one hour 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 488nm or with a UV table operating at 302nm. Excellent sequence coverage in subsequent mass spectrometry analysis of proteolytic peptides is also achieved with this detection method.3

Two-dimensional gel electrophoresis in proteomics: Past, present and future

“…As to image production, most often made now by protein stains, it has made enormous gains in reproducibility over the more than 30 years period in which scientists have run 2D gels. Modern detection methods, such as fluorescent stains [95,96] colloidal Coomassie blue [97], and even modern silver staining [98], where development goes to an end-point, all show a modal coefficient of variation (CV) of ca. 20% (including the variation of the 2D gel process).…”

The Whereabouts of 2D Gels in Quantitative Proteomics

“…While the fixing schemes offer no real advantage over the classical non covalent probes, the nondenaturing schemes offer distinct advantages such as speed, blotting ability [60] [61] and more importantly a very good sequence coverage in subsequent mass spectrometry [61].…”

Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloid cell lines nuclear proteomes

“…Destaining of the spots was carried out by the ferricyanide-thiosulfate method [20] on the same day as silver staining to improve sequence coverage in the mass spectrometry analysis [21]. In some cases, to maximize sequence coverage and avoid the artefacts associated with silver staining, the ultrafast carbocyanine fluorescent stain was used [22].…”