title: A cost-effective method for simultaneous homo-oligomeric size determination and monodispersity conditions for membrane proteins.
abstract: The use of blue native polyacrylamide gel electrophoresis (BN-PAGE) has been reported in the literature to retain both water-soluble and membrane protein complexes in their native hetero-oligomeric state and to determine the molecular weight of membrane proteins. However, membrane proteins show abnormal mobility when compared with water-soluble markers. Although one could use membrane proteins as markers or apply a conversion factor to the observed molecular weight to account for the bound Coomassie blue dye, when one just wants to assess homo-oligomeric size, these methods appear to be too time-consuming or might not be generally applicable. Here, during detergent screening studies to identify the best detergent for achieving a monodisperse sample, we observed that under certain conditions membrane proteins tend to form ladders of increasing oligomeric size. Although the ladders themselves contain no indication of which band represents the correct oligomeric size, they provide a scale that can be compared with a single band, representing the native homo-oligomeric size, obtained in other conditions of the screen. We show that this approach works for three membrane proteins: CorA (42 kDa), aquaporin Z (25 kDa), and small hydrophobic (SH) protein from respiratory syncytial virus (8 kDa). In addition, polydispersity results and identification of the most suitable detergent correlate optimally not only with size exclusion chromatography (SEC) but also with results from sedimentation velocity and equilibrium experiments. Because it involves minute quantities of sample and detergent, this method can be used in high-throughput approaches as a low-cost technique.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore.
authors.forename: Siok Wan
authors.initials: SW
authors.lastname: Gan
authors.forename: Ardcharaporn
authors.initials: A
authors.lastname: Vararattanavech
authors.forename: Nurhuda
authors.initials: N
authors.lastname: Nordin
authors.forename: Said
authors.initials: S
authors.lastname: Eshaghi
authors.forename: Jaume
authors.initials: J
authors.lastname: Torres
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000191:economics* / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry*; D008970:Molecular Weight; D010316:Particle Size; D012995:Solubility
chemical_list: D008565:Membrane Proteins