This work was supported by Wellcome Trust grant 088419 to MDB.

1. Purificación de la GTPasa GST-etiquetados <ol><li> Cultura una E. cepa de E. coli tales como BL21 transformadas con pGEX-Rac1 O / N a 37 ° C, agitando a 220 rpm, en 500 ml de medio de autoinducción (25 mM Na 2 HPO 4, 25 mM KH 2 PO 4, 50 mM NH 4 Cl, 5 mM Na 2 SO 4, 2 mM MgSO 4, 2 mM CaCl 2, 0,5% de glicerol, 0,05% de glucosa, 0,2% de lactosa, 5 g de triptona, 2,5 g extracto de levadura, 100 mg / ml de ampicilina). </li><li> Cosecha bacterias por centrifugación durante 10 min a 10.000 xg, 4 ° C. </li><li> Resuspender sedimento bacteriano en 20 ml de reactivo de extracción de proteínas, inhibidor de la proteasa 1x y se incuba durante 20 min a TA con inversión. </li><li> Aclarar el lisado por centrifugación a 40.000 xg durante 30 min. </li><li> Añadir perlas magnéticas 2 ml de glutatión, se lavaron con solución salina tamponada con fosfato (PBS: 10 mM Na 2 HPO 4, 1,8 mM KH 2 PO 4, NaCl 137 mM, KCl 2,7 mM). </li><li> Incubar durante 2 horas, mezclando por inversión a 4 ° C. </li><li> Lavar perlas de proteína cargada por cuatro veces con 10 ml de PBS, usando un clasificador de partículas magnéticas para precipitar las perlas en cada paso. </li><li> Resuspender perlas en 2 ml de PBS y tienda de proteína cargada-a -80 ° C en 100 ml de alícuotas hasta que se necesite. </li></ol> 2. Expresión de proteínas de unión a GTPasa- <ol><li> El día antes del experimento, los plásmidos que codifican transfectar proteína fluorescente verde (GFP)-etiquetados versiones de cada proteína de unión GTPasa en una separada de 75 cm 2 frasco de HEK293T como sigue. Para la validación de la carga de nucleótidos, marcado con GFP transfectar TrioD1 en una tercera de 75 cm 2 frasco de HEK293T. <ol><li> Diluir polyethylamine a 1 mg / ml en 100 l estéril NaCl 150 mM. </li><li> Añadir 27 l polyethylamine diluido a 223 l de suero reducido de medios de comunicación. </li><li> Añadir 12 g de ADN plasmídico a 250 l de suero reducido de medios de comunicación. </li><li> Incubar cada tubo durante 2 minutos a RT. </li&gt; <li> Combinar el polyethylamine y el ADN se mezcla en un solo tubo y agitar durante 2 min. </li><li> Incubar durante 15 a 20 minutos a temperatura ambiente. </li><li> Reemplazar los medios de crecimiento (medio Eagle modificado por Dulbecco, 10% de suero bovino fetal, 2 mM L-glutamina, sin antibióticos) en 90% confluentes HEK293T con 5 ml de medio de crecimiento fresco. </li><li> Añadir la mezcla de polyethylamine / ADN combinado al matraz y se incuba O / N a 37 ° C, 5% de CO 2. </li></ol></li></ol> 3. Purificación de proteínas de unión a GTPasa- <ol><li> Enjuague frascos de células transfectadas en PBS y drene matraz durante 5 minutos, aspirar líquido libre. </li><li> Raspe células en 500 l de tampón de lisis (50 mM Tris-HCl (pH 7,8), 1% Nonidet P-40, 1x inhibidor de la proteasa) en tubo de microcentrífuga. </li><li> Lisar células mediante la mezcla por inversión a 4 ° C durante 30 min. </li><li> Durante la lisis, lavar dos lotes de 40 mu l perlas de GFP-TRAP tres veces con tampón de lisis fresco, perlas sedimentan en 2700 xg durante 2 min entre lavados. </li><li> Aclarar los lisados ​​por centrifugación a 21.000 xg durante 10 min. </li><li> Transferencia aclaró lisado de cada una de las proteínas de la competencia para separar las perlas lavadas GFP-trampa y permiten proteínas GFP-fusión que se unen durante 2 horas, mezclando por inversión a 4 ° C. Mantenga lisado de células GFP-TrioD1 en hielo. </li><li> Lavar perlas de GFP-TRAP cargados dos veces en 50 mM Tris-HCl (pH 7,8), NaCl 50 mM, 0,7% (w / v) de Nonidet P-40 y dos veces en 50 mM Tris-HCl (pH 7,6), mM MgCl 20 2 , sedimentación cuentas a 2700 xg durante 2 minutos entre lavados. </li><li> Eluir proteínas de fusión GFP-mediante la adición de 40 l 0,2 M glicina (pH 2,5) y pipeteando arriba y abajo durante 30 segundos. Cuentas inmediatamente los sedimentos a 21.000 xg durante 60 s y traslade el líquido a un nuevo tubo de microcentrífuga que contiene 4 l 1 M Tris-HCl (pH 10,4). Haga esto rápidamente para limitar el daño a la proteína purificada. </li><li> Analizar 1 l de cada proteína purificada por Western blot y la sonda con un anticuerpo anti-GFP para establecer rendimiento relativo usando un blott cuantitativasistema ing según el protocolo del fabricante. Alternativamente, determinar las concentraciones de proteínas por ácido (BCA) de ensayo bicinconínico pero esto introduce errores si las proteínas no reaccionan con el ensayo de una manera idéntica o hay proteínas contaminantes. </li><li> Igualar la concentración de proteínas molar mediante la adición de 50 mM Tris-HCl (pH 7,6), 20 mM de MgCl 2. </li></ol> 4. nucleótidos carga de GTPasa <ol><li> Descongelar una alícuota de perlas magnéticas GST-Rac1, preparado en el paso 1. </li><li> Tomar 90 l de perlas de GST-Rac1 y lavar tres veces con 20 mM Tris-HCl (pH 7,6), NaCl 25 mM, DTT 0,1 mM, EDTA 4 mM, usando un clasificador de partículas magnéticas para precipitar las perlas en cada paso. </li><li> Tampón Aspirar a partir de perlas y añadir 100 l 20 mM Tris-HCl (pH 7,6), NaCl 25 mM, DTT 0,1 mM, EDTA 4 mM. </li><li> De acuerdo a si el PIB, GTP o ninguna carga de nucleótidos se requiere para el experimento de competición, añadir 12 l PIB 100 mM, 12 mM l gu 10anosine 5 &#39;- [γ-tio] trifosfato (GTP? S) o no nucleótidos a 60 mu l perlas de GST-Rac1. </li><li> Para los controles de nucleótidos de carga, dividir las perlas restantes en tres alícuotas de 10-mu l y añadir 2 l PIB mM 100, 2 l 10 mM GTP? S o ninguna de nucleótidos a cada tubo. </li><li> Incubar las mezclas de perlas durante 30 minutos a 30 ° C con agitación. </li><li> Estabilizar Rac1 de nucleótidos unida por la adición de 1 M MgCl 2: 3 l a la mezcla experimental (paso 4,4), 0,5 mu l a cada una de las mezclas de control (paso 4.5). </li></ol> 5. Competencia vinculante. <ol><li> Configure 6 tubos de microcentrífuga, conteniendo cada uno: 200 l 50 mM Tris-HCl (pH 7,6), 20 mM de MgCl 2 10 l experimentales cuentas Rac1 nucleótidos cargadas (desde el paso 4.7) Proteína 5 l Rac1 vinculante A (proteína de unión constante) </li><li> A cada tubo, añadir unión Rac1-0, 1, 2,5, 5, 10 o 20 l de proteína B (proteína de unión variable). Estos volúmenes asumen unaproximadamente pueden necesitar ser ajustado concentraciones madre iguales de las proteínas de unión constantes y variables y. <ol><li> Ajuste el volumen de proteínas de unión A y B si hay grandes diferencias en las afinidades de unión de las dos proteínas y esto debe determinarse empíricamente a través de las repeticiones experimentales. Completar el volumen total de la mezcla de unión a 235 l mediante la adición de 50 mM Tris-HCl (pH 7,6), 20 mM MgCl 2. </li></ol></li><li> Establecer un tubo de microcentrífuga que contiene: 200 l 50 mM Tris-HCl (pH 7,6), 20 mM de MgCl 2 10 l experimentales cuentas Rac1 nucleótidos cargadas (desde el paso 4.7) Proteína 10 l Rac1 vinculante A (proteína de unión constante) </li><li> Configure el PIB, GTPyS y no hay tubos de control de nucleótidos: 200 l 50 mM Tris-HCl (pH 7,6), 20 mM de MgCl 2 10 cuentas de Rac1 de control mu l cargados en el paso 4.5 con el PIB, GTPyS o ninguna de nucleótidos y se estabilizaron en el paso 4.7. 180 l HLisado EK293T GFP-TrioD1, preparado como en el paso 3.6 4 l 1 M de MgCl 2 </li><li> Incubar la mezcla durante 2 horas, mezclando por inversión a 4 ° C. </li><li> Lavar las perlas tres veces con 50 mM Tris-HCl (pH 7,6), 20 mM de MgCl 2. </li><li> Eluir con destino proteínas en 20 l tampón de muestra reductor (50 mM Tris-HCl (pH 7), 5% SDS, 20% de glicerol, 0,02 mg / ml de azul de bromofenol, 5% β-mercaptoetanol). </li></ol> 6. Análisis de la competencia <ol><li> Resolver 10 l de la proteína unida (paso 5.6) por electroforesis en gel de poliacrilamida sodio dodecil sulfato (SDS-PAGE) y Western blot. </li><li> Incubar la membrana a 4 ° CO / N en anticuerpo anti-GFP diluyó 1/1000 en tampón de bloqueo a 1x diluyó en PBS, 0,1% de Tween-20 para detectar tanto de las proteínas de unión a GTPasa-etiquetados. </li><li> Se lava la membrana tres veces durante 10 min con PBS, 0,1% de Tween-20. </li><li> Incubar la membrana durante 30 min a TA en DyLight 800 sec-conjugado anti-conejoanticuerpo secundaria, se diluyó 1 / 10.000 en tampón de bloqueo a 1x diluyó en PBS, 0,1% de Tween-20. </li><li> Se lava la membrana tres veces durante 10 min con PBS, 0,1% de Tween-20. </li><li> Analiza la membrana usando un sistema de imagen de infrarrojos, utilizando el software para medir la intensidad de la banda de acuerdo con el protocolo del fabricante. </li><li> Trazar la intensidad de la banda de cada proteína en función del volumen del competidor variable (proteína B). </li><li> Divida el volumen de competidor variable al punto en el que las líneas se cruzan por el volumen de competidor constante (Proteína A, 5 l) para determinar la relación de competidor a la cual se alcanza el equilibrio. </li><li> Para la validación de la condición de nucleótidos de carga, las membranas de la sonda de p21-quinasa activada 1 (PAK1) (un efector) y GFP-TrioD1 (un FMAM), como se describe en los pasos 6.1-6.6. </li></ol>

<ol>
	<li>Burridge, K., Rho Wennerberg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=and+Rac+take+center+stage.">and Rac take center stage.</a> Cell. 116 (2), 167-179 (2004).</li><li>Raftopoulou, M., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+Rho+GTPases+lead+the+way.">Cell migration: Rho GTPases lead the way.</a> Dev Biol. 265 (1), 23-32 (2004).</li><li>Rossman, K. L., Der, C. J., Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GEF+means+go:+turning+on+RHO+GTPases+with+guanine+nucleotide-exchange+factors.">GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors.</a> Nat Rev Mol Cell Biol. 6 (2), 167-180 (2005).</li><li>Worthylake, D. K., Rossman, K. L., Crystal Sondek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=structure+of+Rac1+in+complex+with+the+guanine+nucleotide+exchange+region+of+Tiam1.">structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.</a> Nature. 408 (6813), 682-688 (2000).</li><li>Scheffzek, K., Ahmadian, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPase+activating+proteins:+structural+and+functional+insights+18+years+after+discovery.">GTPase activating proteins: structural and functional insights 18 years after discovery.</a> Cell Mol Life Sci. 62 (24), 3014-3038 (2005).</li><li>Del Pozo, ., A, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+regulate+GTP-Rac+localized+effector+interactions+through+dissociation+of+Rho-GDI.">Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI.</a> Nat Cell Biol. 4 (3), 232-239 (2002).</li><li>Williamson, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin-1C+and+RCC2+guide+mesenchymal+migration+by+trafficking+Rac1+and+controlling+GEF+exposure.">Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure.</a> J Cell Sci. 127 (Pt 19), 4292-4307 (2014).</li><li>Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+the+extracellular+matrix+regulates+the+coupling+of+the+small+GTPase+Rac+to+its+effector+PAK.">Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK.</a> Embo J. 19 (9), 2008-2014 (2000).</li><li>Van Rijssel, J., Hoogenboezem, M., Wester, L., Hordijk, P. L., Van Buul, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-terminal+DH-PH+domain+of+Trio+induces+cell+spreading+and+migration+by+regulating+lamellipodia+dynamics+in+a+Rac1-dependent+fashion.">The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion.</a> PLoS. 7 (1), e29912 (2012).</li><li>Self, A. J., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intrinsic+nucleotide+exchange+and+GTP+hydrolysis+rates.">Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates.</a> Methods Enzymol. 256, 67-76 (1995).</li></ol>

Los autores no tienen nada que revelar.

This protocol describes a method for comparing the relative affinities of pairs of small GTPase-binding proteins. The key steps are the preparation of purified GTPase-binding proteins and the nucleotide loading of the GTPase. The use of GTPase-binding proteins with the same GFP tag, allows the concentrations at which similar amounts of each competitor binds to be accurately determined. The use of recombinant nucleotide-loaded GTPase allows interrogation of the binding properties of the GTPase under specific activity conditions. This step is also the most sensitive as nucleotides will both hydrolyze and detach from the GTPase if the magnesium conditions are not maintained precisely.
In the cell, the large number of GTPase-binding proteins combined with the rapid nucleotide turnover makes such pathways difficult to interpret. The simplicity of this method in comparing only pairs of binding proteins and using carefully controlled nucleotide-loading conditions allows signaling pathways to be elucidated. However, the greatest strength of the protocol is also the greatest weakness as it is a simplification of the in vivo situation. Competition assays can be used to build a robust hypothesis, but this should then be tested in cells by knockdown experiments.
There are three features that must be considered when selecting the GFP-tagged GTPase-binding proteins to be used in the experiment. First, the fusion proteins must express well in mammalian cells, such as HEK293T, as competition assays require a reasonable amount of protein. Second, it must be possible to purify the recombinant protein without significant degradation, and where this is not possible, cloning of a GTPase-binding fragment should be considered. Third, the two GTPase-binding proteins must resolve from one another on SDS-PAGE to allow analysis in section 6.
There are a number of potential caveats to the experiment that need to be considered, and possibly addressed:
Possible denaturation of purified GTPase-binding proteins during the acid elution step or steric hindrance by the GFP tag. In our hands, these have not been a problem, but must be tested. The purified proteins can be tested in functional assays 10. Commercial kits now exist for testing the activity of GEFs or GAPs without the need for isotope-labeled nucleotides. Sequestering proteins, by their nature protect GTPases from GEF or GAP activity, so can be used as competitive inhibitors in the commercial GEF or GAP assays, as we did in our recent publication 7. The relevant feature of proteins that traffic GTPase are the capacity to bind the GTPase, and this can be tested easily in a pull down assay. An alternative approach to testing protein integrity that is applicable to all binding proteins is to titrate protein eluted from GFP-trap beads with glycine with the same protein removed from GFP-trap beads by enzymatic cleavage. The experiment would be analyzed by probing both the GFP-tagged and cleaved protein with an antibody against the protein itself. If the protein is undamaged by elution, equilibrium should be achieved at a 1:1 ratio. This approach would also indicate whether the presence of the GFP tag itself compromises the binding properties of the candidate protein, though this does require the production of a construct with an enzymatic cleavage site between the tag and the binding protein. Whether the protein is compromised by the tag or the elution step, the problem could be addressed by modifying the protocol to use an alternative purification method. Rather than GFP, binding proteins could be His-tagged, purified using Ni-NTA and analyzed using an antibody against the His-tag. The important feature is that both binding proteins must share a common tag although, if necessary, two tags could be added to a protein, one for purification and the other for detection.
The protocol is designed to investigate competition between interactions with the switch I/II domains. Although the majority of GTPase interactions are mediated by this motif, there are some exceptions, most notably the interactions of GDIs that bind to the prenyl tail, as well as obscuring the switch domains. In principle, the protocol could be adapted to use GTPase purified from mammalian cells, so that the GTPase is prenylated, however, the presence of multiple binding sites or allosteric effects complicate the interpretation of competition-binding data. Further problems associated with such a modification are that GDIs co-purify with GTPase from mammalian cells, compromising the purity of the isolated proteins and the hydrophobic nature of the prenyl groups means that prenylated GTPases are associated with either GDI or lipid membrane and such factors would need to be considered in the experiment.
The amount of GST-Rac1 being used in the assay. The constant GTPase binding protein must be at a greater concentration than the Rac1, or when the competitor is added, it will simply bind to free Rac1. It will be immediately obvious if this has happened as binding of the competitor, without a loss of the constant protein, will be detected in much the same way as when the two competing proteins bind to one another as shown in Figure 3B. As an additional control (Step 5.3), a binding reaction containing double the amount of constant binding protein and no variable binding protein should be included (Step 5.3). If the Rac1 in the titration experiment is saturated, doubling the amount of constant binding protein will have no effect on the output. The volumes suggested in the protocol should be appropriate, but the amount of Rac1 can be easily reduced. If binding of the competitor without loss of the constant binding partner is observed, reducing the amount of Rac1 should be attempted before trying to map binding sites to avoid ternary complex formation.
Non-specific interaction of GTPase-binding proteins with the GST or bead, as well as specifically with Rac1. This problem would be manifested by residual binding of the constant GTPase-binding protein, even when the variable GTPase-binding protein has reached a plateau at high concentration. Identification of this issue will be aided by conducting reciprocal experiments where the constant and variable GTPase-binding proteins are swapped. Reciprocal experiments will also greatly improve the accuracy of the estimate of equilibrium point, so should always be included. In cases of non-specific binding, the relative concentrations at which equilibrium is achieved can still be calculated by comparing band intensity between the maxima and minima for each protein, or by measuring the extent of non-specific binding by using GST beads as bait, rather than GST-Rac1.
Pull down assays using different nucleotide-loading conditions should be used to complement the competition assay described in this protocol. Determining the nucleotide preference of partners is important for both understanding the competition events and understanding the signaling pathway that the GTPase-binding protein is involved in. In Figure 2B we analyze binding of proteins with established preference for GTP-loaded or nucleotide-free GTPase as a means to validate nucleotide loading. However, it is sensible to investigate the effect of nucleotide loading on each of the competitors as well. If the hypothetical competitors show different preferences, competition will make less of a contribution to the signaling pathway, and indeed nucleotide turnover is likely to be the mechanism that directs exchange of the binding proteins.

The actin cytoskeleton that determines the shape, polarity and migratory properties of mammalian cells is regulated by the Rho-family of small GTPases. The Rho-family GTPases include RhoA that stimulates cytoskeletal contraction, Rac1 that stimulates actin branching and membrane protrusion, and Cdc42 that has similar effects on actin polymerization to Rac1 and causes the formation of filopodia 1,2. GTPase signaling activity is determined by binding of a nucleotide, which controls the contraction and relaxation of the switch I and switch II loops that mediate the protein-protein interactions with both regulators and effectors. Guanosine 5&#8217;-triphosphate (GTP)-bound GTPases activate downstream effectors, whereas the Guanosine 5&#8217;-diphosphate (GDP)-bound form is inactive. In the cell, cycles of GTP hydrolysis and nucleotide exchange allow rapid turnover of GTPase signals that are necessary for cytoskeletal dynamics. Nucleotide turnover is regulated by three mechanisms. Guanine nucleotide exchange factors (GEFs) stabilize the nucleotide-free GTPase, catalyzing exchange of GDP for GTP, and thereby stimulating GTPase signaling activity 3,4. GTPase-activating proteins (GAPs) catalyze hydrolysis of GTP to GDP, thereby inhibiting GTPase signaling activity 5. Sequestering molecules such as regulator of chromatin condensation 2 (RCC2) and guanine nucleotide dissociation inhibitors (GDIs) obscure the switch loops and in the case of GDIs remove the GTPase from the membrane by interaction with the prenyl tail 6,7. Each of the three classes of regulatory molecule interact with the switch loops, as do the downstream effectors and some trafficking regulators such as coronin-1C 7. The purpose of this protocol is to measure competition for the switch I/II binding site between putative regulators and downstream signaling molecules. It should be noted that competition assays test binding to a shared binding site, so that this protocol is not suitable for testing interactions with other sites, such as binding of GDIs to the prenyl tail.
The subtlety of the conformation differences between active and inactive forms, combined with the labile nature of the bound nucleotide, has made study of GTPase-binding events difficult. The role of the bound nucleotide means that conventional binding assays such as immunoprecipitation or surface plasmon resonance are not well suited to investigation, as the nucleotide cannot be controlled. This obstacle is compounded by the overlap in the binding sites of GEFs, GAPs, effectors, sequestering molecules and trafficking molecules, which make binding data for a single interaction difficult to interpret in the context of the competition that will occur in the cell. Immunoprecipitation, in particular, is compromised by competition between binding partners, as under certain cellular conditions, one binding partner might be identified at the expense of all others, while under other conditions, another partner might dominate. The dynamic nature of GTPase signaling is essential to GTPase function and must be considered when analyzing the relationships between the binding interactions of different regulators. Indeed, we recently described a pathway that relied heavily on competitive binding. We identified coronin-1C as a trafficking molecule that bound to the switch loops of GDP-Rac1 7. In areas of low GEF activity, trafficking would dominate, removing Rac1 from those regions. However, when Rac1 is delivered to regions of the cell where GEF activity is high, the GEF would outcompete coronin-1C, thereby both activating Rac1 and preventing coronin-1C-mediated removal of Rac1 from that area. The model goes further, because the action of the GEF exchanges bound GDP for GTP, shifting the equilibrium still further from coronin-1C. Consequently, Rac1 activity could be explained entirely in terms of competition and relative affinity.
In this protocol, we describe a method for comparing the relative affinities of different binding partners for small GTPases, using Rac1 as an example. By using a purified protein approach, it is possible to piece together a chain of signaling events by pair wise comparison, in an experiment where the bound nucleotide can be closely controlled.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bugbuster</td>
			<td>Novagen</td>
			<td>70584-3</td>
			<td></td>
		</tr>
		<tr>
			<td>COMPLETE protease inhibitor</td>
			<td>Roche</td>
			<td>05 056 489 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione magnetic beads</td>
			<td>Pierce</td>
			<td align="right">88821</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched, average Mw ~25,000</td>
			<td>Sigma Aldrich</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>OPIMEM</td>
			<td>Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10270-1-6</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Trap_A</td>
			<td>Chromotec</td>
			<td>gta-20</td>
			<td></td>
		</tr>
		<tr>
			<td>GDP</td>
			<td>Sigma Aldrich</td>
			<td>G7127</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>GTP&#947;S</td>
			<td>Sigma Aldrich</td>
			<td>G8634</td>
			<td>Highly unstable. Aliquot and store at -80 immediately upon reconstritution</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>Sigma Aldrich</td>
			<td>B6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Living Colors</td>
			<td align="right">632592</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>DyLight 800 conjugated goat anti-rabbit secondary antibody</td>
			<td>Fisher Scientific</td>
			<td align="right">10733944</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAK1 antibody</td>
			<td>Cell Signaling</td>
			<td>2602S</td>
			<td>Use at 1/1000 dilution</td>
		</tr>
		<tr>
			<td>Odyssey SA Infrared Imaging System</td>
			<td>Li-cor</td>
			<td>9260-11PC</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparing the affinity of gtpase-binding proteins using competition assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

Este protocolo está diseñado para calcular las afinidades relativas de los socios de unión para Rac1, sin la necesidad de conocer la concentración exacta de los competidores (Figura 1). Determinación de la concentración de proteína introduce errores y cuando se considera la competencia entre las moléculas en una vía de señalización no es necesario. Sin embargo, es importante saber que los dos competidores tienen la misma concentración molar en las soluciones madre para permitir relaciones simples para ser calculada cuando la adición de diferentes volúmenes para el ensayo. 40 l de perlas de GFP-TRAP tienen una capacidad de unión de ~ 300 pmol por lo que un confluente 75 cm 2 frascos de altamente células que expresan saturará los granos, con el resultado de que las preparaciones de las dos proteínas de unión diferentes serán similares antes del ajuste (Figura 2A). Si una de las proteínas expresa pobremente, este problema se puede superar mediante la purificación de que la proteína de más de un matraz de células. <p class = &quot;jove_content&quot;&gt; La unión de la mayoría de los efectores GTPasa y reguladores depende de la nucleótido-carga de la GTPasa cebo, por lo que es importante para probar si la carga ha sido exitosa. La carga puede ser verificada mediante la precipitación de las proteínas de unión conocida a partir de lisados ​​celulares. Proteínas efectoras, tales como PAK1 se unen a GTP-Rac1 y pueden ser fácilmente precipitaron a partir de lisados ​​y se detectaron por transferencia Western 8 (Figura 2B). GEFs unirse preferentemente al nucleótido libre de GTPasa para estabilizar el estado de transición. Como GEFs son de baja abundancia, por lo general inactivo y con frecuencia blot mal, es mejor que sobreexpresan un FMAM o fragmento del FMAM a prueba GTPasa nucleótidos libres. Con frecuencia usamos la primera homología Dbl de Trio, expresado como una fusión GFP (GFP-TrioD1 9) (Figura 2B) pero cualquier FMAM iba a funcionar. Las proteínas que se unen a la GTPasa PIB cargadas son más raros. Recientemente hemos informado de rcc2 como una de esas proteínas 7, o el PIB de carga puede ser validado simplemente como binding a ni GEF ni efector. La salida del experimento será una transferencia Western que representa las dos parejas de unión GFP-etiquetados unidos a la GTPasa. Mediante el uso de un único anticuerpo para detectar ambas proteínas, las concentraciones a las que cantidades similares de ambos competidores se unen pueden ser determinados y por lo tanto las afinidades relativas reales. En este ejemplo, la competencia entre el dominio de la hélice de la proteína Rac1 la trata, coronin-1C (Rac1-proteína de unión a A), y la proteína Rac1-secuestro, rcc2 (Rac1-proteína de unión B), se demuestra (Figura 3A). Mediante el uso de un volumen constante de la hélice coronin-1C (5 l), y la adición de volúmenes crecientes de rcc2, podemos ver de la GFP blot que el equilibrio se alcanza a 1,25-2,5 l de rcc2 (asterisco), lo que demuestra que tiene un fuerte rcc2 afinidad por Rac1 que coronin-1C. Mediante la medición de la intensidad de las bandas utilizando transferencia Western cuantitativa, y el trazado de los valores medios para cada competitor, el punto de equilibrio se puede calcular con precisión mediante la identificación de los volúmenes en el que las curvas se cruzan (Figura 3B). Uno de los posibles obstáculos para un ensayo de competición éxito es si las parejas de unión se unen entre sí, así como la unión a Rac1. En la Figura 3A + B se demuestra la competencia entre rcc2 y el dominio de la hélice de coronin-1C, en lugar de larga duración coronin-1C. La razón para usar el coronin truncada es que coronin-1C también se une rcc2 a través del dominio de la cola. Cuando de larga duración coronin-1C se valora contra rcc2, se detecta la unión de ambas proteínas, debido a la formación ternario complejo, en lugar de la competencia (Figura 3C). Si la competencia se está produciendo, la unión de una proteína aumentará, mientras que la otra disminuye, y total unida GFP-fusión se mantendrá constante. En los casos en que se forma un complejo ternario es necesario truncar una de la proteína de unión a GTPasa de modo que la competITORS ya no interactúan. <img alt="Figura 1" src="/files/ftp_upload/53254/53254fig1.jpg" /> Figura 1. Flujo de trabajo. Representación esquemática del flujo de trabajo para determinar la afinidad de unión a proteínas GTPasa-utilizando ensayos de competición. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53254/53254fig2.jpg" /> Figura 2. Validación de proteínas purificadas. (A) purificada GFP-etiquetados Rac1 proteínas analizadas por Western blot de unión, sondando con anti-GFP para determinar el rendimiento relativo de las dos proteínas. Este tipo de ecualización durante el experimento permite la concentración de las dos proteínas que se puede modificar para que coincidan en el experimento de unión. (B) GDP, GTPyS y ningún nucleótido-cargado GST-Rac1 se incubó con lisado de HEK293T que expresan GFP-TrioD1 y proteínas detectadas por el trazado de PAK1 endógena o sobreexpresado GFP-TrioD1 obligados. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53254/53254fig3.jpg" /> Figura 3. Western blot de proteína en relación vinculante. Salidas Ejemplo de ensayos de unión con la competencia. Cargó-PIB (A) Rac1 se mezcló con 5 l GFP-coronin-1C dominio hélice y el aumento de los volúmenes de las buenas prácticas agrarias-rcc2 se valora en. Por Western Blot proteínas unidas para GFP, problemas con la detección diferencial de las dos proteínas se evitan y la señal de GFP informes de la relación molar entre las dos proteínas de fusión. Los asteriscos indican las relaciones de competencia en eithelado r del punto de equilibrio. (B) Banda intensidades de proteínas de fusión GFP encuadernados de tres experimentos independientes se midieron mediante transferencia Western cuantitativa, usando anticuerpos y promedios secundarios fluoróforo conjugado conspiraron para calcular la cantidad de rcc2 necesario para alcanzar el equilibrio. (C ) Ejemplo de salida de un experimento donde las proteínas de unión a Rac1 se unen entre sí y forman un complejo ternario, en lugar de competir. PIB-cargado Rac1 se mezcló con 5 l GFP-rcc2 y volúmenes crecientes de-GFP coronin-1C de larga duración se titularon. El aumento de la cota GFP-coronin-1C sin pérdida de cota GFP-rcc2 indica la formación del complejo ternario. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Comparing the Affinity of GTPase-binding Proteins using Competition Assays at JoVE.com

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

La comparación de la afinidad de unión a proteínas GTPasa-utilizando ensayos de competición

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

comparing-affinity-gtpase-binding-proteins-using-competition

Research

JoVE Journal

Biology

9.0K vistas.  University of Bristol. This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

Video: Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Investigación

Biología

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

La cristalización de proteínas de membrana en mesofases lipídico

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-Su purificación: A dos pasos Affinity Protocolo Purificación Cediendo largos proteínas purificadas

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

En Vitro agregación ensayos usando hiperfosforilada proteína tau

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

La investigación de las proteínas priónicas, como la extensión y la toxicidad de Uso del Metazoan Organismo modelo<em&gt; C. elegans</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

Identificación de MyoD Interactome Usando Tandem Affinity purificación Acoplada a la Espectrometría de Masas

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...