Improvements and simplifications in in-gel fluorescent detection of proteins using ruthenium II tris-(bathophenanthroline disulfonate): the poor mans fluorescent detection method

Improvements and simplifications in in-gel fluorescent detection of proteins using ruthenium II tris-(bathophenanthroline disulfonate), the poor man’s fluorescent detection method.

Catherine Aude-Garcia, Véronique Collin-Faure, Sylvie Luche, Thierry Rabilloud

CEA-DSV/iRTSV/LBBSI, Biophysique et Biochimie des Systèmes Intégrés, CEA- Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9, France

Université Joseph Fourier, UMR CNRS-CEA-UJF 5092, CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9

CNRS UMR5092, Biochemistry and Biophysics of Integrated Systems, CEA Grenoble, iRTSV/LBBSI, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9

Correspondence to

Thierry Rabilloud, iRTSV/LBBSI CEA-Grenoble, 17 rue des martyrs,
F-38054 GRENOBLE CEDEX 9 Tel (33)-4-38-78-32-12 Fax (33)-4-38-78-44-99 e-mail: Thierry.Rabilloud@ cea.fr

Abstract Fluorescent detection of proteins is a popular method of detection allying sensitivity, linearity and compatibility with mass spectrometry. Among the numerous methods described in the literature, staining with ruthenium II tris(bathophenanthroline disulfonate) is particularly cost-effective, but slightly cumbersome owing to difficulties in the preparation of the complex and complexity of staining protocols. We describe here modifications on both aspects that allow to perform a higher contrast staining and offer a more robust method of complex preparation, thereby maximizing the advantages of the method.

Numerous constraints apply to detection methods used in gel-based proteomics. They should be linear over a wide range, homogeneous from protein to protein, and of course sensitive. In addition to these core features, they should also be mass spectrometry-compatible, user-friendly and cost-effective. Among the variety of methods used for protein detection after gel electrophoresis [1], fluorescent methods offer an interesting compromise, especially for detection linearity [2] and for compatibility with mass spectrometry [3]. Among the wide variety of fluorescent detection methods that have been developed to detect proteins after gel electrophoresis, two have emerged as standards in the field, one using epicocconone [4] (marketed under the trade name deep purple) and one using an undisclosed fluorescent ruthenium complex and marketed under the trade name Sypro Ruby [5, 6]. Almost simultaneously with the description of Sypro Ruby staining, another stain using a published ruthenium complex (ruthenium II tris(bathophenanthroline disulfonate) was described [7] , with minimal interference with mass spectrometry and much improved cost efficiency compared to commercial formulations. However, the sensitivity of this stain was moderate, and a major improvement was published a few years later [8]. This improvement resulted, however, in a much longer staining period, extending to almost two days after the end of electrophoresis, thereby decreasing the overall productivity of the proteomics setup. This is why intermediate formulations were developed, claiming for equal sensitivity and much improved speed and simplicity of staining [9].

Furthermore, all the protocols using ruthenium II tris(bathophenanthroline disulfonate) are plagued by difficulties in the preparation of the complex. Indeed this preparation involves both complex formation and reduction from ruthenium III to ruthenium II, as most water-soluble ruthenium salts are ruthenium III salts. In the published protocols, this reduction was achieved either with hypophosphoric acid and sodium hydroxide, or by ascorbic acid and sodium hydroxide. As both redox couples are pH-sensitive, the control of the extent of reduction is difficult to manage in the standard proteomics or biochemistry laboratory, so that preparation of the complex occasionally fails. We therefore decided to revisit both aspects of staining with ruthenium II tris(bathophenanthroline disulfonate), namely complex preparation on the one hand, and staining protocol on the other hand, aiming at more simplicity, robustness and performance.

Ammonium formate is a weak but interesting reducing agent [10]. Its solutions are naturally close to neutral pH, thereby needing no pH adjustment, but it is effective only at relatively high temperatures and the overall reducing power is weak. In order to prepare an efficient ruthenium complex, we prepared a solution containing 20 mM potassium pentachloroaquo ruthenate (Alfa Aesar), 60 mM bathophenanthroline disulfonate, disodium salt (Aldrich) and 400 mM ammonium formate (available as a 10 M titrated stock solution from Fluka). Reduction of ruthenium III to ruthenium II and simultaneous complex formation was achieved either by refluxing the solution for three days, or incubating it at 95°C in an oven for three days. In the latter case, because of gas evolution during reduction, it was not possible to tightly close the vessel contai