title: Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts.
abstract: SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20-30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE.
authors.affiliation: Division of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8.
authors.forename: Arianna
authors.initials: A
authors.lastname: Rath
authors.forename: Fiona
authors.initials: F
authors.lastname: Cunningham
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D020106:Acrylamide / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D012015:Reference Standards; D055672:Static Electricity
chemical_list: D008565:Membrane Proteins; D020106:Acrylamide
keywords: apparent size; gel mobility; immunoblotting; protein identification; protein migration
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