Reevaluation of the Electrophoretic Migration Behavior of Soluble Globular Proteins in the Native and Detergent-Denatured States in Polyacrylamide Gels

Westerhuis, Willem H.J.; Sturgis, James N.; Niederman, Robert A.

doi:10.1006/abio.2000.4684

Cited by 39 publications

(33 citation statements)

References 33 publications

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“…2A) derived from the R f values determined at 11-18% T (Fig. 1) were linear on gels <18%, although some concavity was observed as T and M r increased (10,(22)(23)(24)(25) (Fig. 2A, Right).…”

Section: Resultsmentioning

confidence: 87%

“…To quantify these increases, nonlinear regression was applied to define the relationships of K r to M r and of log 10 Y 0 to M r . K r was found to be related to M r by a third-order polynomial in each case (25) (Fig. 2B, Upper), whereas the function relating log 10 Y 0 to M r differed for the TM-mimetic and reference proteins (logarithmic vs. polynomial; Fig.…”

Section: Resultsmentioning

confidence: 94%

“…The latter finding presumably arises from a change in the balance of the charge and frictional forces known to underlie Y 0 as M r is increased (22). In certain systems, the relationship of Y 0 to M r among typical reference proteins may be approximated by monotonic or polynomial functions (23); in others, Y 0 values among globular, water-soluble polypeptides are nearly independent of M r (22,(25)(26)(27).…”

Section: Resultsmentioning

confidence: 99%

“…Any factor(s) that alter the molecular size and shape, and/or net charge, of the polypeptide/DS particle are expected to affect SDS/PAGE migration in a complex manner that will depend on both acrylamide concentration and M r . The smaller polypeptides that are typically analyzed on high-percentage gels may be particularly subject to these variations (25). As examples, alterations in DS loading and/or distribution of DS along the polypeptide chain might increase or decrease SDS/PAGE mobility of small polypeptides (e.g., refs.…”

Section: Discussionmentioning

confidence: 99%

“…The logarithm of R f values obtained by SDS/PAGE on at least three individual gels at each T for each TM-mimetic and reference protein in the M r ranges of 3,500-41,000 and 6,000-55,400, respectively, was plotted as a function of T and fit with linear regression to the relationship (21): log 10 R f = log 10 Y 0 − K r T: [1] To ensure that all data conformed to Eq. 1, R f s of TM-mimetics and reference proteins were sequentially excluded from highest to lowest T until the probability of the linear model reached >0.5 in the runs test; R f values of polypeptides ≥30 kDa obtained on gels of 18% T were omitted from regression analysis (25,35). The resulting lines of best fit had R 2 values ranging from 0.920-0.984, significantly nonzero slopes (P < 0.0001), and probabilities of linearity >0.5 (Tables S3 and S4).…”

Section: Methodsmentioning

confidence: 99%

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Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Rath

Cunningham

Deber

2013

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Rath

Cunningham

Deber

2013

Proc. Natl. Acad. Sci. U.S.A.

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Warping two‐dimensional electrophoresis gel images to correct for geometric distortions of the spot pattern

Gustafsson

Blomberg

Rudemo³

2002

Electrophoresis

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A crucial step in two-dimensional gel based protein expression analysis is to match spots in different gel images that correspond to the same protein. It still requires extensive and time-consuming manual interference, although several semiautomatic techniques exist. Geometric distortion of the protein patterns inherent to the electrophoresis procedure is one of the main causes of these difficulties. An image warping method to reduce this problem is presented. A warping is a function that deforms images by mapping between image domains. The method proceeds in two steps. Firstly, a simple physicochemical model is formulated and applied for warping of each gel image to correct for what might be one of the main causes of the distortions: current leakage across the sides during the second-dimensional electrophoresis. Secondly, the images are automatically aligned by maximizing a penalized likelihood criterion. The method is applied to a set of ten gel images showing the radioactively labeled proteome of yeast Saccharomyces cerevisiae during normal and steady-state saline growth. The improvement in matching when given the warped images instead of the original ones is exemplified by a comparison within a commercially available software.

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Electrophoresis

2016

Electrophoresis in Practice

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Reevaluation of the Electrophoretic Migration Behavior of Soluble Globular Proteins in the Native and Detergent-Denatured States in Polyacrylamide Gels

Cited by 39 publications

References 33 publications

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

Warping two‐dimensional electrophoresis gel images to correct for geometric distortions of the spot pattern

Electrophoresis

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