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- Malm, Christer, et al.
(author)
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Evaluation of 2-D DIGE for skeletal muscle: Protocol and repeatability
- 2008
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In: The Scandinavian Journal of Clinical & Laboratory Investigation. - : Informa Healthcare. - 0036-5513 .- 1502-7686. ; 68:8, s. 793-800
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Journal article (peer-reviewed)abstract
- Proteomic analysis has the potential to yield vast amounts of data. The available proteomic methods have been hampered by methodological errors in quantification due to large gel-to-gel variations. The inclusion of an internal standard greatly reduces this variation, and therefore the purpose of this investigation was: 1) to develop a sample preparation protocol for human skeletal muscle for two-dimensional differentiated gel electrophoresis (DIGE) and 2) to investigate the repeatability of one particular system, the Ettan™ DIGE. To test repeatability, nine aliquots from the same homogenate were labelled with three different CyDye™ dyes (Cy2, Cy3, Cy5). Samples were run on 1824 cm gels, scanned with a Typhoon™ 9410 laser scanner and analysed in the DeCyder™ software. When selecting spots appearing only in triplicate (n = 1314), the mean error was 1.7 % (SD: 10.5 %; 95 % CI: 1.1-2.4 %). When setting the significance level to 99 %, no false-positive changes in protein volume ratios were detected. In the protocol presented here, only 0.5 mg tissue was used and separation of >2500 distinct protein spots in the pH range 3-11 and MW 10-200 kDa. Changes in protein abundance of <20 % could be detected. The method is especially useful when comparing muscle proteins between different conditions; for example, healthy and diseased tissue, before and after treatment or different exercise protocols.
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