Proteomic capacity of recent fluorescent dyes for protein staining.
Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to both the number of proteins analysed, the accuracy of spot quantification and reproducibility. In this work, we compared the efficiency of recent fluorophores to stain Arabidopsis total protein extract: Sypro Ruby (SR), Deep Purple (DP) and 5-hexadecanoylamino-fluorescein (C16-F). In addition, classical visible dyes, colloidal Coomassie blue (CCB) and silver nitrate (SN), were also included. High quality images were obtained for the three fluorescent dyes, DP giving the cleaner background, whereas spikes were observed with SR and a rough background with C16-F. On the other hand, saturation occurred for abundant spots with SR and DP. For a same protein load the number of detected spots ranged between 250 for CCB and 800 for SR in the sequence SR > DP approximately SN > C16-F > CCB. These differences were shown to rely mainly on the sensitivity between dyes leading to the detection of additional spots belonging to classes of lower abundance. Analysis of the distribution of variation coefficients for spots from replicates showed differences in the staining reproducibility between dyes that ranged in the order SR > C16-F > DP > SN > CCB. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.
Chevalier
François
F
Laboratoire de Protéomique, INRA, UR 1199, 2 place Viala, 34060 Montpellier Cedex 1, France.
Rofidal
Valérie
V
Vanova
Pavlina
P
Bergoin
Alexis
A
Rossignol
Michel
M
D017360:Arabidopsis / Q000737:chemistry; D029681:Arabidopsis Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D040901:Proteomics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*
D029681:Arabidopsis Proteins; D005456:Fluorescent Dyes