title: Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two-dimensional gel electrophoresis for proteomic analysis.
abstract: Performing 2-DE of lipid-rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2-D-proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2-DE profiling of myelin proteins is very important for the detection of immuno-reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent-mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB-14-4 resulted in significant increase in protein representation of myelin in 2-DE compared with the addition of ASB-14-4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB-14-4 strategy than other strategies of myelin sample processing such as ASB-14-4 to myelin or ethanol or acetone or methanol-ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2-D gel is achieved.
authors.affiliation: Amrita Center for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences, Amrita Viswavidyapeetham, Kochi, Kerala, India.
authors.forename: Sreepriya
authors.initials: S
authors.lastname: Nair
authors.forename: Tessy
authors.initials: T
authors.lastname: Xavier
authors.forename: Madathiparambil Kumaran Satheesh
authors.initials: MK
authors.lastname: Kumar
authors.forename: Sharmistha
authors.initials: S
authors.lastname: Saha
authors.forename: Krishnakumar N
authors.initials: KN
authors.lastname: Menon
mesh_terms: D000476:Alkanesulfonates; D000818:Animals; D001923:Brain Chemistry; D011232:Chemical Precipitation; D003902:Detergents / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000431:Ethanol; D000432:Methanol; D009185:Myelin Proteins / Q000737:chemistry* / Q000302:isolation & purification; D009186:Myelin Sheath / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D013816:Thermodynamics
chemical_list: D000476:Alkanesulfonates; D003902:Detergents; D009185:Myelin Proteins; D020543:Proteome; D000431:Ethanol; D000432:Methanol