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 watersoluble 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 membranespanning 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. gel mobility | protein migration | protein identification | apparent size | immunoblotting L aemmli's system for polyacrylamide gel protein electrophoresis in the presence of the detergent SDS (SDS/PAGE) is one of the most cited methodological papers in life sciences (1). The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, resulting in "overnight" adoption of the technique in biochemistry, cell biology, immunology, and virology (2). Considered "the single most useful analytical tool to study protein molecules" (3), SDS/ PAGE is routinely used for simultaneous determination of protein heterogeneity and molecular weight in applications ranging from diagnosis of hereditary red cell membrane disorders to evaluation of recombinant protein expression and purification procedures. Protein analysis by SDS/PAGE is relatively simple, affordable, and rapid (4): A buffer containing a tracking dye and SDS is added to the sample of interest, the mixture is applied to a polyacrylamide gel, and a potential difference is used to drive the dye and the resulting anionic particle composed of protein and dodecyl sulfate (DS) through the gel. The distance traveled by the protein/DS particle from the top of the gel is then divided by that of the dye to obtain relative migration (R f ), and molecular weight [as relative molecular mass (M r )] determined by comparison of this value with a logarithmic plot derived from the R f s and M r s of reference proteins.Fractionation on SDS/PAGE is contro...

Expression, purification, and preliminary characterization of human presenilin-2

“…4a, inset), the higher Mw band appeared reproducibly at ∼100 kDa. This type of Mw shift for helical membrane proteins dependent on the acrylamide concentration has been previously studied and reported [64]. At ∼50 kDa, two diffuse bands were observed on 10% SDS-PAGE (Fig.…”

One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent

“…1B). The apparent Mw was smaller compared with its theoretical Mw and this anomalous migration appears common for membrane proteins [21,22]. The AqpZ proteins were further identified by mass spectrometry.…”

An overview of technical considerations for Western blotting applications to physiological research

“…Alternatively gradient gels (e.g. 4–12%) provide uniform resolution across the molecular weight spectrum (Rath et al 2013). Other gel types such as agarose may be used, but are less common as they are predominantly used for very large molecular weight proteins (e.g.…”