title: High-resolution Native-PAGE for membrane proteins capable of fluorescence detection and hydrodynamic state evaluation.
abstract: An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins.
authors.affiliation: Molecular Signaling Research Team, Structural Physiology Research Group, RIKEN SPring-8 Center, Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ihara
authors.forename: Noriko
authors.initials: N
authors.lastname: Matsuura
authors.forename: Atsuko
authors.initials: A
authors.lastname: Yamashita
mesh_terms: D002850:Chromatography, Gel; D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057446:Hydrodynamics; D008565:Membrane Proteins / Q000302:isolation & purification*
chemical_list: D008565:Membrane Proteins; D004143:Dioctyl Sulfosuccinic Acid; D049452:Green Fluorescent Proteins