Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines

TL;DR

This paper introduces a rapid, sensitive, and nonfixing fluorescent protein staining method using oxacarbocyanine dyes during electrophoresis, compatible with downstream mass spectrometry analysis.

Contribution

It presents a novel coelectrophoretic staining technique with carbocyanine dyes that is fast, sensitive, nonfixing, and suitable for proteomics applications.

Findings

Detection within 1 hour after electrophoresis

Sensitivity between colloidal CBB and ruthenium complexes

Compatible with subsequent MS analysis

Abstract

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 fluroescent 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.