Evaluation of 2-D DIGE for skeletal muscle: Protocol and repeatability

Malm, ChristerUmeå universitet,Institutionen för integrativ medicinsk biologi (IMB),Department of Intergrative Medical Biology, Section for Anatomy, Umeå University, Sweden; Winternet, Boden, Sweden (författare)

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.

Hadrevi, JennyHögskolan i Gävle,Umeå universitet,Idrottsmedicin,Centrum för belastningsskadeforskning,Umeå University, Department of Integrative Medical Biology, Section for Anatomy, Umeå, Sweden(Swepub:hig)jhi (författare)
Bergström, Sven-AndersUmeå universitet,Medicin,Department of Public Health and Clinical Medicine, Umeå University, Sweden(Swepub:umu)svsbem99 (författare)
Pedrosa-Domellof, FatimaUmeå universitet,Institutionen för integrativ medicinsk biologi (IMB),Oftalmiatrik,Department of Integrative Medical Biology, Section for Anatomy, Umeå University(Swepub:umu)fape0001 (författare)
Antti, HenrikUmeå universitet,Kemiska institutionen,Department of Chemistry, Section for Organic Chemistry, Umeå University, Sweden(Swepub:umu)hean0004 (författare)
Svensson, MichaelUmeå universitet,Idrottsmedicin,Department of Surgical and Preoperative Sciences, Section for Sports Medicine, Umeå University, Sweden(Swepub:umu)misv0002 (författare)
Frängsmyr, LarsUmeå universitet,Institutionen för integrativ medicinsk biologi (IMB),Department of Intergrative Medical Biology, Section for Anatomy, Umeå University, Sweden and Department of Surgical and Preoperative Sciences, Section for Sports Medicine, Umeå University, Sweden(Swepub:umu)lacfrr79 (författare)
Umeå universitetInstitutionen för integrativ medicinsk biologi (IMB) (creator_code:org_t)

Ingår i:The Scandinavian Journal of Clinical & Laboratory Investigation: Informa Healthcare68:8, s. 793-8000036-55131502-7686

Av författaren/redakt...: Malm, Christer; Hadrevi, Jenny; Bergström, Sven- ...; Pedrosa-Domellof ...; Antti, Henrik; Svensson, Michae ...; visa fler...; Frängsmyr, Lars; visa färre...

Om ämnet

MEDICIN OCH HÄLSOVETENSKAP: MEDICIN OCH HÄLS ...; och Klinisk medicin; och Oftalmologi

MEDICIN OCH HÄLSOVETENSKAP: MEDICIN OCH HÄLS ...; och Medicinska och f ...; och Cell och molekyl ...

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