High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore.
This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple.
Tokarski
Caroline
C
Laboratory of Organic and Macromolecular Chemistry, UMR CNRS 8009 and Proteomics, Post-Translational Modifications and Glycobiology, IFR 118, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Cren-Olivé
Cécile
C
Fillet
Marianne
M
Rolando
Christian
C
D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009942:Organometallic Compounds / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*
D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D011506:Proteins; C511353:bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succinimidyl ester