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

Arianna Ratha, Fiona Cunninghama,b,1, and Charles M. Debera,b,2

aDivision of Molecular Structure and Function, Research Institute, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; and bDepartment of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8

Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved August 16, 2013 (received for review June 13, 2013) SDS/PAGE is universally used in biochemistry, cell biology, and (5)]. Larger particles become trapped within the gel meshwork immunology to resolve minute protein amounts readily from and migrate slower than smaller species. Low-percentage gels tissue and cell extracts. Although molecular weights of water- are therefore typically used to resolve larger proteins, and vice soluble proteins are reliably determined from their SDS/PAGE versa. Acrylamide concentrations compatible with routine use mobility, most helical membrane proteins, which comprise 20–30% are usually from 4–20% T due to practical considerations, of the human genome and the majority of drug targets, migrate to because gels outside of this range are too fragile or too brittle, positions that have for decades been unpredictably slower or respectively, to withstand the physical manipulation(s) required faster than their actual formula weight, often confounding their for protein visualization and/or immunoblotting. identification. Using de novo designed transmembrane-mimetic Most globular, water-soluble proteins are reliably identified by polypeptides that match the composition of helical membrane- their SDS/PAGE mobility relative to corresponding reference spanning sequences, we quantitate anomalous SDS/PAGE frac- proteins typically used to estimate molecular weight. However, tionation of helical membrane proteins by comparing the relative this group of well-behaved polypeptides does not include helical mobilities of these polypeptides with typical water-soluble refer- transmembrane (TM) proteins, macromolecules that comprise fi ence proteins on Laemmli gels. We nd that both the net charge 20–30% of the human genome (6), comprise the majority of drug and effective molecular size of the migrating particles of trans- targets (7), and are the focus of major pharmaceutical discovery membrane-mimetic species exceed those of the corresponding ref- efforts (8). For example, the first true G protein-coupled re- erence proteins and that gel acrylamide concentration dictates the ceptor to be determined to high resolution, 39-kDa bovine impact of these two factors on the direction and magnitude of rhodopsin (9), migrates on SDS/PAGE to positions consistent anomalous migration. Algorithms we derived from these data with sizes as low as 30 kDa (10). In fact, we have previously compensate for this differential effect of acrylamide concentration shown that the gel mobility of helical TM proteins seldom cor- on the SDS/PAGE mobility of a variety of natural membrane pro- responds to formula molecular weight (11). This phenomenon of teins. Our results provide a unique means to predict anomalous “anomalous migration” can arise as a consequence of the high migration of membrane proteins, thereby facilitating straightfor- hydrophobicity and concomitant binding of DS by TM proteins ward determination of their molecular weights via SDS/PAGE. at levels that exceed those of water-soluble polypeptides (12). fi However, quantitation of DS binding stoichiometry is not rou- gel mobility | protein migration | protein identi cation | apparent size | fi immunoblotting tine and consumes milligram amounts of puri ed samples. Thus, the impact of enhanced DS binding on the direction and mag- ’ nitude of anomalous migration has remained unpredictable for aemmli s system for polyacrylamide gel protein electropho- decades, with helical TM proteins variously exhibiting gel mo- Lresis in the presence of the detergent SDS (SDS/PAGE) is bility reduced, equivalent, or increased relative to reference one of the most cited methodological papers in life sciences (1). proteins (11–13). Such differences are generally disregarded when The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, Significance 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/ SDS/PAGE is a protein analysis technique universally used in PAGE is routinely used for simultaneous determination of protein biochemistry, cell biology, immunology, and virology, where heterogeneity and molecular weight in applications ranging from proteins are separated by size on a gel matrix of polyacrylamide. However, most helical membrane proteins, which diagnosis of hereditary red cell membrane disorders to evaluation – of recombinant protein expression and purification procedures. are biomolecules that comprise 20 30% of genomes and the Protein analysis by SDS/PAGE is relatively simple, affordable, and majority of drug targets, migrate to positions on SDS/PAGE that have for decades been unpredictably larger or smaller rapid (4): A buffer containing a tracking dye and SDS is added to than their actual size. We have found that the magnitude and the sample of interest, the mixture is applied to a polyacrylamide direction of migration among membrane protein mimetics are gel, and a potential difference is used to drive the dye and the controlled by the acrylamide concentration in the gel. Our resulting anionic particle composed of protein and dodecyl sulfate results facilitate straightforward SDS/PAGE analysis of these (DS) through the gel. The distance traveled by the protein/DS important biomolecules. particle from the top of the gel is then divided by that of the dye

to obtain relative migration (Rf), and molecular weight [as Author contributions: A.R. and C.M.D. designed research; A.R. and F.C. performed re- relative molecular mass (Mr)] determined by comparison of this search; A.R. contributed new reagents/analytic tools; A.R. and C.M.D. analyzed data; and A.R. and C.M.D. wrote the paper. value with a logarithmic plot derived from the RfsandMrsof reference proteins. The authors declare no conﬂict of interest. Fractionation on SDS/PAGE is controlled by the molecular This article is a PNAS Direct Submission. size and shape of the protein/DS particle, its net charge, and the 1Present address: Mitacs, York University, Toronto, ON, Canada M3J 1P3. accessible spaces among the acrylamide ﬁbers that comprise 2To whom correspondence should be addressed. E-mail: [email protected]. the gel matrix as determined by the total concentration of acryl- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. amide and bis-acrylamide cross-linker [T; Materials and Methods 1073/pnas.1311305110/-/DCSupplemental.

15668–15673 | PNAS | September 24, 2013 | vol. 110 | no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1311305110 Downloaded by guest on September 28, 2021 protein identity is known or can be confirmed in orthogonal the Tris-glycine system) were selected for the present work molecular weight determination procedures but, in many in- because they are used in the majority of determinations of pro- stances, raise questions of protein folding, oligomeric organiza- tein complexity and molecular weight (16), and are used most tion, proteolytic processing, posttranslational modification(s), often among reported systems in our database of helical TM alternative splicing, antibody cross-reaction, and/or degradation. protein SDS/PAGE mobilities (11) (SI Materials and Methods). These issues become acute in SDS/PAGE analyses of tissue or Gels using these buffers at >10% T resolve reference proteins in cell extracts, where reasonable molecular weight estimates re- the range of ≥14–200 kDa, whereas those with ≥14% T frac- main crucial for protein identification. tionate species ≥3.5 kDa (Mark12 Unstained Standard on Life Here, we quantitate anomalous SDS/PAGE fractionation of Technologies Novex Tris-Glycine Gel SDS/PAGE Migration helical membrane proteins by comparing the relative mobilities Charts, www.invitrogen.com). Given that our TM-mimetics en- of de novo designed TM-mimetic peptide polymers with typical compass the range from 3.5–41 kDa (13), we accordingly chose water-soluble reference proteins on Laemmli gels ranging from to analyze SDS/PAGE migration behavior on gels of 11–18% T, 11–18% T. We find that net charge and effective molecular size in 1% T intervals. among the migrating TM-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide con- Gel Mobility of TM-Mimetics Relative to Reference Proteins Changes centration dictates the impact of these two factors on the direction with Acrylamide Concentration. The set of TM protein mimetics and magnitude of anomalous migration. Algorithms derived from we designed and prepared are polymers of a peptide with the “ ” these data compensate for the differential effect of acrylamide core sequence NH2-SKSKS-Leu20-SKSKS-NH2, termed pL20 concentration on the SDS/PAGE mobility of a variety of natural (13). The average length, high hydrophobicity, and abundance of membrane proteins. Our results provide a straightforward means Leu in natural membrane-spanning regions are recapitulated in to predict anomalous migration of membrane proteins relative to the 20-Leu segment of pL20, whereas its basic Lys and hydro- reference polypeptides, facilitating their identification by molec- philic Ser residues resemble the native termini of such sequences ular weight in SDS/PAGE applications. (17). Sequence patterns that mediate oligomerization (e.g., refs. 18–20) were specifically excluded from the design. CD spectra of Results pL20 and its derivatives were characteristic of stable helical A variety of buffer systems for SDS/PAGE have been described secondary structure (Figs. S1 A and B and S2), whereas Förster (e.g., refs. 1, 14, 15). From these, Laemmli buffers (also known as resonance energy transfer between dansyl- and dabsyl-derivatized BIOCHEMISTRY

Fig. 1. TM-mimetics switch migration positions relative to reference proteins on SDS/PAGE at various acrylamide concentrations. Representative gels in- −3 dicating the migration position and Mr × 10 of the apolar peptide polymers comprising our TM-mimetics (Left lanes), the globular water-soluble polypeptides comprising a commercial blend of reference proteins (Right lanes), and the migration of the bromophenol blue tracking dye (far right of each set of gels) are shown. T is given at the top of each gel image. The sharpness of bromophenol blue may reﬂect the moving boundaries known to occur in SDS/PAGE (36, 37).

Rathetal. PNAS | September 24, 2013 | vol. 110 | no. 39 | 15669 Downloaded by guest on September 28, 2021 pL20 could not be detected in SDS (Fig. S1 C–E), confirming its quantify these increases, nonlinear regression was applied to monomeric state. Nevertheless, the 3.5-kDa TM-mimetic corre- define the relationships of Kr to Mr and of log10 Y0 to Mr. Kr was sponding to pL20 migrated as an apparent dimer of ∼7 kDa at found to be related to Mr by a third-order polynomial in each ≥14% T (Fig. 1), whereas TM-mimetics greater than or equal to case (25) (Fig. 2B, Upper), whereas the function relating log10 Y0 ∼30 kDa had enhanced mobility on 11–13% gels (Fig. 1). In- to Mr differed for the TM-mimetic and reference proteins (log- deed, each TM-mimetic less than or equal to ∼18 kDa migrated arithmic vs. polynomial; Fig. 2B, Lower). The latter finding slower than reference proteins of comparable size (Fig. 1), presumably arises from a change in the balance of the charge and whereas those greater than or equal to ∼30 kDa changed gel shift frictional forces known to underlie Y0 as Mr is increased (22). In direction from migration faster to slower than the reference certain systems, the relationship of Y0 to Mr among typical ref- proteins as T was raised (Fig. 1). Plots relating gel mobility (Rf) erence proteins may be approximated by monotonic or polynomial to the logarithm of molecular weight (Mr) confirmed that the functions (23); in others, Y0 values among globular, water-soluble SDS/PAGE mobility of TM-mimetics relative to reference pro- polypeptides are nearly independent of Mr (22, 25–27). teins was reduced at smaller Mrs, equivalent at an Mr corresponding to the intersection point of the two curves, and increased as Mrs Predicting Differential Gel Mobility from Gel Acrylamide Concentration, exceeded the intersection point (Fig. S3 and Table S1). Molecular Size, and Net Charge. Relationships of Kr and of log10 Y0 to Mr were used to derive equations that predict anomalous migration Molecular Size and Net Charge Are Larger Among TM-Mimetics than in terms of the difference in gel mobility of a TM-mimetic(s) vs. Reference Proteins. Having excluded the possibility of self-assembly reference protein(s), expressed as a fraction of the distance traveled as the origin of these phenomena, we used the procedure first by the tracking dye (Fig. 3 A and B). These equations show that gel proposed by Ferguson (21) and summarized by Chrambach and mobility among the TM-mimetics is variously equal, reduced, or Rodbard (5) to measure two parameters of protein/DS com- increased relative to reference proteins as a function of Mr and T plexes that together contribute to migration on SDS/PAGE: (Fig. 3 A and B). As illustrated schematically in Fig. 3C, a 30-kDa retardation coefficient (Kr), a measure of effective molecular size TM-mimetic will migrate equivalently to a water-soluble reference that is determined by the total mass and shape of the migrating protein of the same size on SDS/PAGE at 13% acrylamide. particle, and free electrophoretic mobility (as log10 Y0; Materials However, the same TM-mimetic will migrate anomalously slow on and Methods), a descriptor of net charge. Both quantities are a 20% gel, with mobility reduced by 3.3% of the distance traveled measured by determining mobility on gels of various acrylamide by the tracking dye. Conversely, at 9.0% acrylamide, the 30-kDa concentrations and generating a Ferguson plot of log10 Rf vs. T TM-mimetic will migrate faster than a 30-kDa reference protein, that is linear with slope −Kr and y-intercept log10 Y0 (Eq. 1). with mobility increased by 15% of the distance traveled by the Ferguson plots (Fig. 2A) derived from the Rf values determined tracking dye. at 11–18% T (Fig. 1) were linear on gels <18%, although some When we tested predictions made by our equations with a concavity was observed as T and Mr increased (10, 22–25) (Fig. database of helical TM protein SDS/PAGE mobilities (11), we 2A, Right). The linear regression analysis used to obtain Kr and found that gel acrylamide concentration was the predominant log10 Y0 therefore eliminated Rf of ≥30-kDa polypeptides at factor dictating the direction and magnitude of anomalous mi- 18% T. Kr and log10 Y0 among the TM-mimetics were larger gration among 16 (73%) of 22 of these proteins (Eq. S9, Dataset than those of the reference proteins (Fig. 2B), an outcome S1, and Fig. S4). This level of accuracy is remarkable, given that consistent with the enhanced DS loading capacity expected for the SDS/PAGE experiments in the database were performed by sequences of enhanced hydrophobicity (12) (Table S2). To various groups over 20 y and were not filtered for bis-acrylamide

Fig. 2. Molecular size and net charge of TM-mimetics are larger than those of reference proteins. (A) Ferguson plots of TM-mimetics (n = 5–8, ○)and

reference proteins (n = 5–8, ●), grouped by molecular weight for clarity of presentation. Points represent the average of at least three independent Rf measurements and error bars ± SD. The best-fit line to each TM-mimetic (solid lines) and reference protein (dashed lines) is shown (R2 > 0.9, P < 0.0001; Tables −3 S3 and S4). Mr × 10 is indicated at the right of the best-fit line; bracketed Arabic or Roman numerals indicate the degree of polymerization of the TM- mimetic (13) or the identity of the reference protein (Table S2), respectively. (B) Values of Kr (Upper) and log10 Y0 (Lower), obtained from the best-fit lines shown in A, are plotted vs. Mr. Points corresponding to the TM-mimetics (n = 11, ○) and reference proteins (n = 6, ●) are shown ± fit errors, labeled according to the scheme in A. Lines connecting Kr and log10 Y0 values of TM-mimetics (solid lines) and reference proteins (dashed lines) were obtained by nonlinear regression (R2 > 0.9).

15670 | www.pnas.org/cgi/doi/10.1073/pnas.1311305110 Rath et al. Downloaded by guest on September 28, 2021 concentration, an important determinant of Kr (5). Given that each TM-mimetic has a fixed number of 10 flanking polar residues per TM sequence, whereas the extramembrane regions of natural helical TM proteins may encompass a few to hundreds of residues, it seemed reasonable that the six exceptions might be distinguished by the proportion of extramembrane vs. TM sequence. The formula Mr per TM domain among the outliers was nevertheless statistically indistinguishable from those with cor- rectly predicted gel mobility (Dataset S1). However, we note that proteins with large extramembrane regions are generally excluded from the Mr range of the present data. It thus remains possible that the SDS/PAGE mobility of the TM-mimetics may more closely resemble natural membrane proteins with short loops than that of proteins with large extramembrane domains. Discussion Anomalous SDS/PAGE mobility of helical membrane proteins thus arises as a direct consequence of enhanced net charge and effective molecular size among their protein/DS particles relative to the particles of water-soluble reference proteins. The outcome of this difference on gel mobility, however, is tied to the interplay between these two quantities, polypeptide molecular weight, and the space available within the acrylamide gel matrix [as determined by T (5)]. Thus, it may be surmised that on gels of acrylamide concentration lower than equivalent gel mobility (Teq), increased net charge dominates Rf, resulting in increased mobility. However, as acrylamide concentration increases, migration becomes increasingly impeded by larger particle size, resulting in a gradual reduction in gel mobility, and ultimately a “switch” to reduced mobility once Teq is exceeded. Trends of increased mobility among larger helical membrane proteins and of reduced mobility among smaller helical membrane proteins (11) may therefore arise from using low- or high-percentage acrylamide gels, respectively, for SDS/PAGE analysis. 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 Mr. The smaller polypeptides that are typically analyzed on high-percentage gels may be par- ticularly 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. 12, 28, 29), depending on the acrylamide concentration of the gel and the buffer system used for analysis. Indeed, differential protein/DS contacts among folded vs. un- folded β-barrel membrane proteins may underlie variation in their gel shifts (30). Quantitation of the observed pivotal role of acrylamide concentration using the algorithm shown in Fig. 3 should nevertheless facilitate interpretation of any SDS/PAGE application where an accurate molecular weight is required (i.e., identifica- BIOCHEMISTRY tion of helical membrane proteins can be accomplished with water-soluble reference proteins when gels as close as possible to Teq are used). However, the dependence of Kr on the electrophoresis buffer system (31, 32) and on cross-linker concentration (32, 33) means that the algorithms presented here will be most

Fig. 3. SDS/PAGE mobility shifts of TM-mimetics relative to reference pro-

teins. (A and B) Mobility shift is expressed as a fraction of the distance range is not typically applied to low-Mr proteins. (C) Quantitative cartoon of traveled by the tracking dye (ΔRf = Rf, TM − Rf, Ref; SI Materials and Methods). anomalous SDS/PAGE migration calculated for a 30-kDa TM-mimetic (TM, Positive and negative values of ΔRf (green and red, respectively) indicate white) vs. a 30-kDa water-soluble reference protein (Sol, black). Gels (gray increased or reduced mobility of TM-mimetics relative to reference proteins. rectangles) are arranged in order of increasing total acrylamide concentra-

A ΔRf of zero (bold contour) indicates equivalent mobility. Graphs are tion, with the mobility of the tracking dye (dye, blue arrow) shown at the far shaded according to mobility shift, with darker colors corresponding to right. At 9.0% T, mobility of the TM-mimetic (green arrow) is increased by

regions of larger magnitude migration differences. (B) Contour map of the 15% of the distance traveled by the dye (ΔRf of +0.15). At 13% T, the TM- data presented in A, viewed along the ΔRf axis (a 90° rotation toward the mimetic migrates equivalently to the water-soluble protein (black arrow, reader). We caution that mobility shifts calculated for 4–10% T represent ΔRf of zero; asterisk in B). At 20% T, mobility of the same TM-mimetic (red extrapolations of present data; in practice, this acrylamide concentration arrow) is reduced by 3.3% of the distance traveled by the dye (ΔRf of −0.033).

Rathetal. PNAS | September 24, 2013 | vol. 110 | no. 39 | 15671 Downloaded by guest on September 28, 2021 accurate when applied to Laemmli gels at the bis-acrylamide in parallel to at least three individual gels at each T. Electrophoresis was concentration used here (Materials and Methods). With this in performed at 125 V in Tris-glycine SDS running buffer [25 mM Tris base, 192 mind, we note that anomalous SDS/PAGE migration among TM mM glycine, 0.1% (wt/vol) SDS, pH 8.3] at room temperature for 100 min – proteins should disappear if TM-mimetics are used as size (11 14% T) or until the bromophenol blue tracking dye was near the gel end – standards (13); in applications where protein(s) are unknown, (15 18% T). Gels were removed from the cassettes and immediately imaged with visible light to record the positions of the tracking dye and the pre- such reagents may prove to be essential. stained marker proteins. After visible imaging, gels were silver-stained using Materials and Methods the SilverXpress Silver Staining Kit (Life Technologies) according to the manufacturer’s directions. Gel images taken before and after staining were Production of TM-Mimetics. The TM-mimetics were produced from peptides aligned based on the positions of the prestained marker proteins. The dis- with the sequence H2N-Cys-SKSKS-Leu20-SKSKS-Cys-NH2 as previously de- tances of each polypeptide band and of the bromophenol blue dye from the scribed (13). Briefly, purified peptide was self-polymerized at the sulfhydryl moiety of Cys in a “one-pot” reaction with a bis-maleimidoethane (BMOE) top of the separating gel were measured and divided to obtain relative cross-linker in an aqueous 80% (vol/vol) 2,2,2-trifluoroethanol solution mobility (Rf). buffered at pH 7.5 with 20 mM Tris. BMOE was selected as a conjugating linkage because of the sulfhydryl specificity of its reaction at neutral pH, the Ferguson Plots. The logarithm of Rf values obtained by SDS/PAGE on at least stability of the resulting thioether linkage to changes in pH and to reducing three individual gels at each T for each TM-mimetic and reference protein in – – agents, the trans-configuration of linkages, and the commonality of thio- the Mr ranges of 3,500 41,000 and 6,000 55,400, respectively, was plotted as ether conjugates in biomaterials. a function of T and fit with linear regression to the relationship (21):

log R = log Y − K T: [1] SDS/PAGE. Gels were cast in disposable minigel 8-cm × 8-cm, 1.0-mm-thick 10 f 10 0 r cassettes (Life Technologies) using mixtures containing appropriate volumes To ensure that all data conformed to Eq. 1,Rs of TM-mimetics and reference ′ f of a 40% T stock solution [29:1 acrylamide/N,N -methylenebis(acrylamide), proteins were sequentially excluded from highest to lowest T until the 3.3% C; BioRad], degassed ultrapure water, ultrapure SDS (BioUltra; Fluka), probability of the linear model reached >0.5 in the runs test; Rf values of and stacking (0.375 M Tris·HCl, pH 6.8) or separating (1 M Tris, pH 8.8) polypeptides ≥30 kDa obtained on gels of 18% T were omitted from re- buffers, where T [%, (wt/vol)] = [g acrylamide + g N,N′-methylenebis(acryl- gression analysis (25, 35). The resulting lines of best fithadR2 values ranging amide)] per 100 mL of solution and C [%, (wt/vol)] = 100 × [g N,N′-methyl- from 0.920–0.984, significantly nonzero slopes (P < 0.0001), and probabilities enebis(acrylamide)] per 100 mL of solution/T (34). The polymerization catalysts of linearity >0.5 (Tables S3 and S4). K and log Y were obtained from the N,N,N′,N′-tetramethylethylenediamine (TEMED; BioRad) and freshly pre- r 10 0 absolute slope and y-intercept of these best-fit lines, and we have opted pared 5% (wt/vol) ammonium persulfate (APS; BioRad) in ultrapure water here to compare log Y values in lieu of transforming to Y . This modifi- were added immediately before casting. Separating gels were 6 cm in length 10 0 0 cation facilitates derivation of the equations that predict the SDS/PAGE and contained 11–18% T, 0.375 M Tris (pH 8.8), 0.1% (wt/vol) SDS, 0.025% mobility of TM-mimetics relative to reference proteins (Eqs. S3–S9). (vol/vol) TEMED, and 0.125% (wt/vol) APS; stacking gels were 2 cm in length and contained 4% T, 0.125 M Tris (pH 6.8), 0.1% (wt/vol) SDS, 0.04% (vol/vol) TEMED, and 0.4% (wt/vol) APS. Separating and stacking gels were each Statistical Analysis. Linear regression, nonlinear regression, runs tests, and allowed to polymerize for 1 h at room temperature, after which cassettes statistical analyses were performed using Prism version 4.0a for Macintosh < fi were sealed in packages containing a small volume of ultrapure water and (GraphPad). P values 0.05 were considered signi cant. stored at 4 °C overnight before use. Aliquots of TM-mimetics were dissolved in 1× Tris-glycine SDS sample ACKNOWLEDGMENTS. We thank Prof. Karen Fleming (Johns Hopkins buffer (Life Technologies). Mark12 Unstained Standard (Life Technologies) University) for helpful suggestions regarding the analyses presented in this was diluted 1/20 in 1× Tris-glycine SDS sample buffer before application to manuscript. This work was supported by grants to C.M.D. from the Medical and Related Sciences (MaRS) Innovation Proof of Principle Program (MaRS gels. Five microliters of the TM-mimetic blend and 5 microliters of the di- POP Grant MI-POP 2010-0088); the Natural Sciences and Engineering Re- luted Mark12 Standard were applied to each gel. Ten microliters of SeeBlue search Council Idea-to-Innovation Program (NSERC I2I Grant 411522-10); Pre-Stained Protein Standard (Life Technologies) was applied undiluted to and the Canadian Institutes of Health Research (Grant FRN-5810). The Hos- the first and last lanes of each gel in order to facilitate alignment of images pital for Sick Children owns the intellectual property associated with the before and after staining. TM-mimetics and reference proteins were applied composition and preparation of the TM-mimetic polypeptide reagents.

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