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

GST etiketli GTPaz 1. Saflaştırma <ol><li> Kültür, bir E. örneğin 220 rpm&#39;de çalkalanarak 37 ° C&#39;de pGEX-Rac1 O / N ile transforme BL21 gibi E. coli soyundan, autoinduction ortam 500 ml (25 mM Na-2 HPO 4, 25 mM KH 2 PO 4, 50 mM NH4CI, 5 SO 4 mM Na 2, 2 mM MgSO 4, 2 mM CaCl2,% 0.5 gliserol,% 0.05 Glukoz,% 0.2 laktoz, 5 g tripton, 2.5 g maya ekstresi, 100 ug / ml Ampisilin). </li><li> 10.000 x g&#39;de, 4 ° C&#39;de 10 dakika için santrifüjleme ile hasat bakteri. </li><li> 20 ml protein ekstre edici reaktif, 1 x proteaz inhibitörü bakteriyel pelet yeniden süspanse edin ve inversiyon ile oda sıcaklığında 20 dakika inkübe edilir. </li><li> 30 dakika boyunca 40.000 x g&#39;de santrifüjleme ile lizat açıklığa kavuşturulması. </li><li> Fosfat tamponlu tuz ile yıkandı, 2 ml glutasyon manyetik boncuklar, ekleme (PBS: 10 mM Na-2 HPO 4, 1.8 mM KH 2 PO 4, 137 mM NaCI, 2.7 mM KCI). </li><li> 4 ° C&#39;de ters çevirme ile karıştırılması, 2 saat süre ile inkübe edilir. </li><li> Her adımda boncuk çökelmesi için bir manyetik parçacık ayırıcıyla, 10 mi, PBS ile protein yüklü boncuk dört kez yıkayın. </li><li> Gerekli olana kadar yeniden süspanse 100 ul alikotları -80 ° C &#39;de 2 ml PBS içinde boncuk ve mağaza protein yüklü. </li></ol> GTPaz bağlama proteinlerinin ekspresyonu 2. <ol><li> Şöyle gün önce deney, yeşil flüoresan proteini (GFP) kodlayan transfekte plazmidler HEK293T ayrı bir 75-cm 2 şişe içine, her GTPaz bağlayıcı proteinin sürümlerini etiketli. Nükleotid yükleme doğrulama için, transfect HEK293T üçüncü 75 cm 2 balona TrioD1 GFP etiketli. <ol><li> 100 ul içinde 1 mg / ml steril 150 mM NaCl polyethylamine seyreltin. </li><li> 223 ul azaltılmış serum medya 27 ul seyreltilmiş polyethylamine ekleyin. </li><li> 250 ul düşük serum ortamı 12 ug plazmit DNA ekleyin. </li><li> Oda sıcaklığında 2 dakika için her bir tüp inkübe edin. </li&gt; <li> Polyethylamine birleştirin ve DNA 2 dakika boyunca tek bir tüp ve vorteks karıştırır. </li><li> Oda sıcaklığında 15-20 dakika kuluçkalayın. </li><li> 5 ml taze büyütme ortamı ile% 90 konfluent HEK293T üzerindeki büyüme ortamı (Dulbecco Modified Eagle Ortam,% 10 fetal bovin serumu, 2 mM L-glutamin, antibiyotikler herhangi bir) değiştirin. </li><li> Şişeye Birleştirilen polyethylamine / DNA karışımı ekleyin ve 37 ° C&#39;de,% 5 CO2 O / N inkübe edin. </li></ol></li></ol> GTPaz bağlama proteinlerinin saflaştırılması 3. <ol><li> PBS içinde transfekte edilmiş hücrelerin şişeler durulayın ve serbest sıvı aspire, 5 dakika boyunca balon boşaltın. </li><li> 500 ul liziz tamponu içinde hücrelerini Scrape (50 mM Tris-HCl (pH 7.8),% 1 Nonidet P-40, 1 x proteaz inhibitörü) mikrofüj tüpüne. </li><li> 30 dakika boyunca 4 ° C &#39;de ters çevirme ile karıştırılarak Hücreleri,. </li><li> Liziz sırasında yıkamalar arasında 2 dakika boyunca 2700 x g&#39;de 40 ul GFP-Trap boncuklar iki çok taze lizis tamponu ile üç kez, çöktürülmesi, boncuk yıkama. </li><li> 10 dakika boyunca 21.000 x g&#39;de santrifüjleme ile lizatları açıklığa kavuşturulması. </li><li> Aktarım 4 ° C de ters çevirme ile karıştırılması, yıkandı, GFP-trap boncuk ayrılması ve GFP-füzyon proteinleri, 2 saat boyunca bağlamasına izin vermek için rakip proteinin her birinin lizat açıklık. Buz üzerinde GFP-TrioD1 hücrelerinden lizat tutun. </li><li> 50 mM Tris-HCl (pH 7.8), 50 mM NaCI içinde iki kez yüklü GFP-Trap boncuk yıkayın,% 0.7 (w / v) Nonidet P-40 ve iki kez de 50 mM Tris-HCI (pH 7.6) içinde, 20 mM MgCl2 , yıkamalar arasında 2 dakika boyunca 2700 x g&#39;de boncuk çökeltildi. </li><li> 40 ul 0.2 M glisin (pH 2.5) ilave etmek ve 30 saniye süreyle aşağı ve yukarı pipetlenerek GFP füzyon proteinlerini Zehir. 4 ul 1 M Tris-HCl (pH 10.4) içeren yeni bir mikro santrifüj tüpüne 21.000 60 s için xg ve transfer sıvısının hemen tortu boncuklar. Saflaştırılmış protein zarar sınırlamak için hızla yapın. </li><li> Kantitatif Blott kullanarak göreceli verim kurmak için, bir anti-GFP antikor ile Western blot ve sonda her bir saflaştırılmış protein 1 ul analizüreticinin protokolüne uygun olarak sistemi olarak konumlandırılır. Seçenek olarak ise, bisinkoninik asit (BCA) deneyi ile protein konsantrasyonlarını belirlemek ancak proteinler, özdeş bir tarzda tahlil ile reaksiyona girmeyen ya da kirletici proteinler vardır, bu hatalar ortaya çıkarır. </li><li> 50 mM Tris-HCI (pH 7.6), 20 mM MgCl2 ilave edilerek mol protein konsantrasyonu dengeleyin. </li></ol> GTPaz 4. nükleotid yükleme <ol><li> Aşama 1 &#39;de hazırlanan GST-Rac1 manyetik boncuk, bir kısım çözülme. </li><li> Her adımda boncuk çökelmesi için bir manyetik parçacık ayırıcıyla GST-Rac1 boncuk 90 ul alın ve 20 mM Tris-HCl (pH 7.6), 25 mM NaCI, 0.1 mM DTT, 4 mM EDTA ile üç kez yıkayın. </li><li> Aspire boncuklardan tamponu ve 100 ul 20 mM Tris-HCl (pH 7.6), 25 mM NaCI, 0.1 mM DTT, 4 mM EDTA ekleyin. </li><li> GDP, GTP veya nükleotid yükleme rekabet deneyinde için gerekli olup olmadığını göre, 12 ul 100 mM GDP, 12 ul 10 mM gu eklemeanosine 5 &#39;- [γ-tiyo] trifosfat (GTP yS) ya da 60 ul GST-Rac1 boncuklar için bir nükleotid. </li><li> Nükleotid yükleme kontrolleri, üç 10-ul kısma kalan boncuk bölünmüş ve 2 ul 100 mM GSYİH, 2 ul 10 mM GTPtS veya her tüpe hiçbir nükleotid ekleyin. </li><li> Çalkalama ile 30 ° C &#39;de 30 dakika boyunca damla karışımları inkübe edin. </li><li> Deney karışımı 3 ul (aşama 4,4), kontrol karışımları (adım 4.5) her bir 0.5 ul: 1 M MgCl2 ilave edilerek nükleotid bağlı Rac1 stabilize. </li></ol> Bağlama 5. Rekabet. <ol><li> 6 mikrofüj tüpleri kurmak, her biri: 200 ul 50 mM Tris-HCl (pH 7.6), 20 mM MgCl2 (Aşama 4,7), 10 ul deney nükleotid yüklü Rac1 boncuklar 5 ul Rac1 bağlayıcı protein A (sabit bağlayıcı protein) </li><li> Her bir tüpe, 0, 1, 2.5, 5, 10 veya 20 ul Rac1 bağlayıcı protein B (değişken bağlayıcı protein) ekleyin. Bu birimler bir varsayıyorumpproximately eşit hisse sabit ve değişken bağlama proteinleri konsantrasyonları ve ayarlanması gerekebilir. <ol><li> Orada iki proteinin bağlama yakınlıkları büyük farklılıklar vardır ve bu deneysel tekrarlar yoluyla ampirik olarak tespit edilmelidir eğer bağlayıcı proteinler A hacimleri ve B ayarlayın. 50 mM Tris-HCI (pH 7.6), 20 mM MgCl2 ilave edilerek 235 ul bağlanma karışımın toplam hacmini hazırlayın. </li></ol></li><li> Içeren bir mikrofuge&#39;de tüp ayarlayın: 200 ul 50 mM Tris-HCl (pH 7.6), 20 mM MgCl2 (Aşama 4,7), 10 ul deney nükleotid yüklü Rac1 boncuklar 10 ul Rac1 bağlayıcı protein A (sabit bağlayıcı protein) </li><li> GSYİH, GTPtS ve hiçbir nükleotid kontrol tüpleri ayarlayın: 200 ul 50 mM Tris-HCl (pH 7.6), 20 mM MgCl2 10 ul kontrol Rac1 boncuk GSYİH, GTPtS veya nükleotid ile adım 4.5 yüklü ve adım 4.7 stabilize. 180 ul HAşama 3.6 gibi hazırlanmıştır EK293T GFP-TrioD1 lizatı, 4 ul 1 M MgCI2 </li><li> 4 ° C&#39;de ters çevirme ile karıştırılması, 2 saat süre ile inkübe karışımı. </li><li> 50 mM Tris-HCI (pH 7.6), 20 mM MgCl2 ile boncuk üç kez yıkayın. </li><li> Elute indirgeyici örnek tamponu 20 ul bağlı proteinleri (50 mM Tris-HCI (pH 7),% 5 SDS,% 20 gliserol, 0.02 mg / ml bromofenol mavi,% 5 β-merkaptoetanol). </li></ol> Yarışmanın 6. Analizi <ol><li> Sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) ve Western blot ile bağlı protein (aşama 5,6) 10 ul giderin. </li><li> 4 ° CO de membran inkübe / N anti-GFP antikorda Bloklama Tamponu içinde 1/1000 oranında seyreltilmiş PBS&#39;de 1x seyreltilmiş,% 0.1 Tween-20 isim levhası GTPaz bağlama proteinlerinin her ikisi de tespit etmek. </li><li> PBS ile 10 dakika,% 0.1 Tween-20, membran üç kez yıkayın. </li><li> 800 konjuge edilmiş anti-tavşan sek DyLight içinde oda sıcaklığında 30 dakika boyunca zarın inkübesekonder antikor Bloklama Tamponunda 1 / 10,000 seyreltilen,% 0.1 Tween-20, PBS içinde 1 x seyreltilmiştir. </li><li> PBS ile 10 dakika,% 0.1 Tween-20, membran üç kez yıkayın. </li><li> Üreticinin protokolüne göre, bant yoğunluğu ölçmek için yazılımını kullanarak, kızılötesi görüntüleme sistemi kullanılarak zar tarama. </li><li> Değişken rakip (Protein B) birim karşı her proteinin bant yoğunluğunu çiziniz. </li><li> Çizgiler, denge elde edildiği rakip oranının saptanması için sabit bir rakip (Protein A, 5 ul) hacimce kesiştiği noktada değişken yarışmacının hacmi bölün. </li><li> Adımda 6,1-6,6 tarif edildiği gibi p21-aktive edilmiş kinaz 1 (PAK1) (bir efektör) ve GFP-TrioD1 (GEF), nükleotid yükleme durumu, prob membran geçerli olabilmesi için. </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>

Yazarlar ifşa hiçbir şey yok.

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.

Bu protokol, rakip hassas konsantrasyonunu bilinmesi gereken (Şekil 1) olmadan Rac1 için bağlayıcı ortaklar nispi afiniteler hesaplamak için tasarlanmıştır. Protein konsantrasyonunun saptanması hatalar oluşturan bir sinyal yolunun, moleküller arasındaki rekabeti söz konusu olduğunda gerekli değildir. Ancak, iki yarışmacı tahlil farklı hacimleri eklerken basit oranları hesaplandı izin vermek için stok solüsyonları aynı molar konsantrasyona sahip olduğunu bilmek önemlidir. GFP-Trap boncuk 40 ul 300 pmol yani birleşik 75 cm yüksek iki farklı bağlama proteinleri preparatları düzenlenmesinden önce benzer olacaktır ve bunun sonucunda, boncuk doyuracak olan ifade eden hücrelerin 2 şişeler ~ bir bağlama kapasitesine sahip (Şekil 2A). Proteinlerin bir zor ifade ederse, bu sorun, hücrelerin birden çok şişeden bu proteinin saflaştırılması ile aşılabilir. <p class = &quot;jove_content&quot;&gt; en GTPaz efektörlerinin ve düzenleyici bağlanma yem GTPaz nükleotid yükleme bağlıdır, bu yüzden yük başarılı olup olmadığını test etmek için önemlidir. Yükleme hücresi lizatları bilinen bağlanma proteinleri çökeltme ile doğrulanabilir. Örneğin GTP Rac1 ve PAK1 bağ olarak efektör proteinler kolayca lizatları çökeltildi ve Western blot 8 (Şekil 2B) ile tespit edilebilir. Geçiş durumunu stabilize etmek GTPaz ücretsiz nükleotid tercihen bağlamak GEFS. GEFS genellikle inaktif düşük bolluk, vardır ve sık sık kötü leke gibi, deney nükleotid serbest GTPaz bir GEF veya GEF fragmanı aşın daha iyidir. Biz sık sık Trio&#39;nun ilk İKO benzerlik kullanmak, bir GFP füzyon (GFP-TrioD1 9) (Şekil 2B) olarak ifade ancak herhangi GEF çalışmaya devam eder. GSYİH yüklü GTPaz bağlanan proteinler nadirdir. Bindi basitçe valide edilebilir böyle bir protein 7 veya GSYİH yükleme gibi Biz son zamanlarda RCC2 bildirdiNe GEF&#39;ten de efektör ng. Deney çıkış GTPaz bağlanan iki GFP etiketli bağlama partnerleri gösteren bir Western blot olacaktır. Her iki proteini tespit etmek için, tek bir antikor kullanarak, her iki rakip içindeki miktarlarda bağlanan hangi konsantrasyonları belirlenebilir ve bu nedenle göreceli afıniteleri anlaşılmaktadır. Rac1 kaçakçılığı protein pervane etki alanı arasındaki bu örnek yarışmada, Koronin-1C (protein A Rac1-bağlama) ve Rac1-ayırıcı protein, RCC2 (protein B Rac1-bağlama), (Şekil 3A) gösterilmiştir. Koronin-1C pervane (5 ul) sabit bir hacim kullanılarak ve RCC2 artan miktarlarda ekleyerek, RCC2 güçlü olduğunu gösteren, GFP arasından bu denge RCC2 (yıldız) 1,25-2,5 ul ulaşılır leke görebilir Koronin-1C daha Rac1 için afinite. Her competito için ortalama değerler kantitatif Batı lekelenmesi kullanılarak bantların yoğunluğunu ölçen ve komplo iler, denge noktası hacimleri hangi eğrileri (Şekil 3B) kesişir belirleyerek doğru hesaplanabilir. Bağlanma eşleri birbirine olarak Rac1 bağlanma ile bağlanması halinde başarılı bir rekabet deneyinde mümkün engellerden biri. Şekil 3A + B biz oldukça tam uzunlukta Koronin-1C daha RCC2 ve Koronin-1C pervane etki alanı arasındaki rekabeti, gösterir. Kesik Koronin kullanılmasının nedeni Koronin-1C, aynı zamanda kuyruk alanı üzerinden RCC2 bağlanması anlamına gelir. Tam uzunlukta Koronin-1C RCC2 karşı titre edildiğinde, her iki proteinin bağlanması nedeniyle üçlü kompleks oluşumu yerine rekabet (Şekil 3C) için tespit edilir. Yarışma oluşup ise, bir proteininin diğer düşüşler olurken artacak ve toplam bağlı GFP-füzyon sabit kalacaktır. Durumlarda üçlü bir kompleksi, GTPaz bağlayıcı proteinin bir kesecek için gerekli olan formları burada böylece competitors artık etkileşim. <img alt="figür 1" src="/files/ftp_upload/53254/53254fig1.jpg" /> Şekil 1. İş Akışı. Yarışma analizleri kullanılarak GTPaz-bağlayıcı proteinlerin afinite belirlemek için iş akışı şematik. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/53254/53254fig2.jpg" /> Saflaştırılmış proteinlerin Şekil 2. Doğrulama. (A) saflaştınldı GFP etiketli iki proteinin nispi verimi belirlemek için, anti-GFP ile sondalama Western blot ile analiz bağlayıcı proteinlerin Rac1. Deney sırasında dengeleme Bu tür da bağlanma deneyinde eşleşecek şekilde iki protein konsantrasyonu ayarlanmasına olanak tanır. (B) GDP, GTPtS ve hiçbir nükleotid yüklü GST-Rac1 endojen PAK1 veya aşırı GFP-TrioD1 için komplo tespit proteinleri GFP-TrioD1 ifade HEK293T gelen lizat ile inkübe ve bağlıydı. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/53254/53254fig3.jpg" /> Görece protein Şekil 3. Western Blot analizi ile yapışma. Örnek çıktıları yarışması bağlayıcı tahlillerinden. (A) Rac1 5 ul GFP Koronin-1C pervaneli alanı ile karıştırılmış ve titre edildi GFP-RCC2, gittikçe artan miktarlarda edildi GDP-yüklendi. Tarafından GFP için Western blotting bağlanmış proteinler, iki proteinin diferansiyel tayinle belirlenmesine ile ilgili sorunlar önlenir ve GFP sinyali iki füzyon proteinleri arasındaki molar oranı bildirir. Yıldız eithe rekabet oranlarını gösterirdenge noktasının r tarafında. (B), üç bağımsız deneyin ilişkili bağlantı GFP füzyon proteinlerinin Bant yoğunlukları, dengeye ulaşmak için gerekli olan fluorofor-konjuge sekonder antikor ve ortalamalar RCC2 miktarını hesaplamak için çizilen kullanılarak niceliksel Western blot ile ölçüldü. (Cı Rac1 bağlayıcı proteinler rekabet etmek yerine birbirine bağlanan ve üçlü bir kompleks oluşturur bir deney) Örnek çıktı. GDP-yüklü Rac1 5 ul GFP RCC2 ile karıştırıldı ve GFP-Koronin-1C tam boyda, gittikçe artan miktarlarda edildi titre edilmiştir. Bağlanmış GFP Koronin-1C&#39;de artış bağlanmış GFP RCC2 kaybı olmadan üçlü kompleks oluşumunu gösterir. <a href="https://www.jove.com/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">Lütfen Bu rakamın büyük halini görmek için buraya tıklayın.</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.

Rekabet tahlilleri kullanılarak GTPaz bağlayıcı proteinlerin afinitesini

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 Görüntüleme.  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.

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

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

Biyoloji

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.

Lipidik Mesophases Membran Proteinleri Kristallendirilmesi

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.

Yüksek verim Kristalizasyon lipidik Bicelle Yöntemiyle Membran Proteinleri

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.

Tandem Affinity Arıtma kullanarak Protein etkileşim Ortaklar belirlenmesi

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.

Affinity etiketli Rekombinant Proteinler Yüksek throughput Arıtma

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-His saflaştırma: Tam boy Saflaştırılmış Proteinler Verim iki aşamalı bir saflaştırma protokolü Affinity

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.

In Vitro toplama Deneyler hiperfosforile tau proteini kullanılarak

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.

Metazoan Model Organizma kullanma Serim ve toksisitesi prion-benzeri Proteinler incelenmesi<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.

Tandem Affinity Arıtma kullanarak MyoD interaktom belirlenmesi kütle spektrometresi ile birleştiğinde

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