Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes

被引:173
作者
Shaw, J
Rowlinson, R
Nickson, J
Stone, T
Sweet, A
Williams, K
Tonge, R
机构
[1] AstraZeneca, Global Prot Sci & Supply Enabling Sci & Technol B, Macclesfield SK10 4TG, Cheshire, England
[2] Amersham Biosci, Prote R&D, Grove Ctr, Amersham, Bucks, England
关键词
fluorescent dyes; two-dimensional difference gel electrophoresis;
D O I
10.1002/pmic.200300439
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye(TM) DIGE Cy(TM)3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels.
引用
收藏
页码:1181 / 1195
页数:15
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