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

1.净化GST标记的GTP酶<ol><li>文化的E.大肠杆菌菌株如大肠杆菌BL21转化的质粒pGEX-Rac1的O / N在37℃下，振荡以220rpm，在500ml自身诱导培养基（25mM的磷酸氢二钠，25mM的KH 2 PO 4，50mM的氯化铵 ， 5mM的用 Na 2 SO 4，2mM的硫酸镁 ，2毫氯化钙 ，0.5％甘油，0.05％葡萄糖，0.2％乳糖，5克蛋白胨，2.5克酵母提取物，100微克/毫升氨苄青霉素）。 </li><li>收获细菌通过离心10分钟以10,000×g离心，4℃。 </li><li>重悬细菌沉淀在20ml蛋白质提取试剂，1×蛋白酶抑制剂孵育20分钟，在室温将其与倒置。 </li><li>澄清裂解液离心以40,000×g离心30分钟。 </li><li>加2ml谷胱甘肽磁珠，用磷酸盐缓冲盐水（PBS:10mM的磷酸氢二钠，1.8毫KH 2 PO 4，137 mM氯化钠，2.7毫米氯化钾）。 </li><li>孵育2小时后，通过倒转在4℃下混合。 </li><li>用10ml PBS洗涤蛋白加载珠四次，使用磁粉分拣以沉淀珠粒在每一个步骤。 </li><li>重悬蛋白质装载于2ml PBS中珠和储存在-80℃下在100μl等份直到需要。 </li></ol> GTP酶结合蛋白2的表达<ol><li>一天实验前，转染的质粒编码绿色荧光蛋白（GFP）-tagged每个GTP酶结合蛋白的版本到一个单独的75-cm 2的烧瓶HEK293T的如下。对于核苷酸装载的验证，转染GFP标记TrioD1到第三个75厘米2瓶HEK293T的。 <ol><li>稀释polyethylamine至1mg / ml，在100μl的无菌150mM的NaCl洗涤。 </li><li>加入27微升稀释polyethylamine至223微升减少血清的培养基。 </li><li>加12微克质粒DNA到250微升减少血清的培养基。 </li><li>孵育各管在室温2分钟。 </LI&gt; <li>结合polyethylamine和在单管中，涡旋2分钟的DNA混合。 </li><li>下室温温育15-20分钟。 </li><li>用5毫升新鲜生长培养基更换生长培养基（Dulbecco氏改良的Eagle介质，10％牛胎儿血清，2mM的L-谷氨酰胺，不含抗生素）上90％汇合的HEK293T。 </li><li>将合并的polyethylamine / DNA混合物添加到该烧瓶中，并培育O / N在37℃，5％ 的 CO 2。 </li></ol></li></ol> 3.纯化GTP酶结合蛋白的<ol><li>冲洗转染的细胞在PBS中的烧瓶和漏极烧瓶5分钟，吸出游离的液体。 </li><li>刮去细胞在500μl裂解缓冲液（50mM的Tris-HCl（pH7.8），1％​​的Nonidet P-40，1×蛋白酶抑制剂）到微量离心管中。 </li><li>裂解细胞通过倒置，在4℃下进行30分钟的混合。 </li><li>在裂解，洗两批40微升GFP-陷阱珠三次新鲜的裂解液，沉淀的珠2,700 XG的洗涤之间2分钟。 </li><li>澄清的裂解物通过离心21,000×g离心10分钟。 </li><li>转印澄清每个参赛者的蛋白质分离洗涤GFP-陷阱珠和允许的GFP融合蛋白结合2小时的裂解物，由倒置，在4℃下混合。保持溶胞产物从GFP-TrioD1细胞在冰上。 </li><li>在50mM的Tris-HCl（pH值7.8），50mM NaCl中洗两次加载GFP-陷阱珠，0.7％（重量/体积）的Nonidet P-40的和两次在50mM的Tris-HCl（pH值7.6），20mM的MgCl 2的，2,700 xg离心洗涤之间2分钟沉降珠。 </li><li>通过加入40微升的0.2M甘氨酸（pH值2.5）和移液器上下吹吸30秒洗脱GFP的融合蛋白。立即沙珠在21000 XG为60秒和转让液含有4微升的1M的Tris-HCl（pH值10.4），一个新的离心管中。做到这一点很快，限制损坏纯化蛋白。 </li><li>分析1微升通过Western印迹和探针用抗GFP抗体每种纯化蛋白的使用定量blott建立相对产量根据制造商的协议的系统上。可替代地，确定的蛋白浓度通过二辛可宁酸（BCA）测定法但这会引入错误，如果蛋白质不与化验反应中以相同的方式或有污染蛋白质。 </li><li>通过加入50mM的Tris-HCl（pH值7.6），20mM的MgCl 2的摩尔相等蛋白质浓度。 </li></ol> GTP酶的4核苷酸装<ol><li>解冻的GST-Rac1的磁珠，在步骤1中制备一个等份。 </li><li>采取90微升的GST-Rac1的珠和洗涤三次用20mM的Tris-HCl（pH为7.6），25 mM氯化钠，0.1mM的DTT，4mM EDTA的，使用磁性粒子分选器以沉淀珠粒在每一个步骤。 </li><li>从珠吸缓冲区，并添加100微升20毫米的Tris-HCl（pH值7.6），25毫米氯化钠，0.1毫米DTT，4毫摩尔EDTA。 </li><li>根据国内生产总值，GTP或没有加载核苷酸是否需要竞争的实验中，加入12微升100毫米的GDP，12微升10毫区anosine 5&#39; - [γ-硫代]三磷酸（GTPγS）或无核苷酸到60微升的GST-Rac1的珠。 </li><li>对于核苷酸负荷控制，分割剩余的珠串成三个10微升等份，并添加2微升100毫米GDP，2微升10毫米GTPγS或无核苷酸，每管。 </li><li>孵育珠混合物30分钟，在30℃下搅拌。 </li><li>稳定通过加入1M MgCl 2的核苷酸结合的Rac1:3μl到实验混合物（步骤4.4），0.5微升至每个控制混合（步骤4.5）的。 </li></ol> 5.竞争结合。 <ol><li>设立了6个离心管，每片含: 200微升的50mM的Tris-HCl（pH值7.6），20mM的MgCl 2的 10微升试验核苷酸加载Rac1的珠（来自步骤4.7） 5微升Rac1的结合蛋白A（常结合蛋白） </li><li>到各管中，加入0,1，2.5，5，10或20微升的Rac1结合蛋白B（可变结合蛋白）。这些卷假设pproximately等于库存浓度恒定和可变结合蛋白的，并可能需要调整。 <ol><li>调节结合蛋白A和卷乙如果在两种蛋白质的结合亲和力大的差异，这应根据经验通过实验重复来确定。补结合混合物与235微升的总体积通过加入50mM的Tris-HCl（pH值7.6），20mM的MgCl 2的。 </li></ol></li><li>建立含有微量离心管: 200微升的50mM的Tris-HCl（pH值7.6），20mM的MgCl 2的 10微升试验核苷酸加载Rac1的珠（来自步骤4.7） 10微升Rac1的结合蛋白A（常结合蛋白） </li><li>成立了国内生产总值，GTPγS无核苷酸对照管: 200微升的50mM的Tris-HCl（pH值7.6），20mM的MgCl 2的 10微升控制Rac1的珠子装入步骤4.5与GDP，GTPγS或无核苷酸和稳定步骤4.7。 180微升水中EK293T GFP-TrioD1裂解物，制备如步骤3.6 4微升1M的氯化镁 </li><li> 2小时孵育该混合物中，通过倒转在4℃下混合。 </li><li>用50mM的Tris-HCl（pH值7.6），20mM的MgCl 2的洗珠三次。 </li><li>洗脱结合的蛋白于20μl还原样品缓冲液（50mM的Tris-HCl（pH 7）中，5％的SDS，20％甘油，0.02毫克/毫升溴酚蓝，5％β巯基乙醇）。 </li></ol>竞争6.分析<ol><li>解决将10μl结合蛋白（步骤5.6）的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western印迹。 </li><li>孵育该膜在4℃的CO / N的抗GFP抗体在封闭缓冲液稀释1/1000稀释于PBS 1倍，0.1％Tween-20的同时检测标记的GTP酶结合蛋白。 </li><li> 10分钟，用PBS，0.1％Tween-20的洗膜三次。 </li><li>在DyLight孵育膜在室温30分钟800缀合的抗兔秒ondary抗体，稀释1 / 10,000中的封闭缓冲液稀释在PBS，0.1％Tween-20的1倍。 </li><li> 10分钟，用PBS，0.1％Tween-20的洗膜三次。 </li><li>扫描使用红外成像系统的膜，使用该软件根据制造商的协议来测量带强度。 </li><li>图中的每个蛋白的带强度对变量的竞争对手（蛋白B）的量。 </li><li>除以变量竞争者的体积在的点上的线相交由恒定竞争者（蛋白A，5微​​升）的容积，以确定在哪些达到平衡的竞争者比率。 </li><li>为了验证核苷酸负荷状态，探测膜的按照步骤6.1-6.6中描述的p21激活激酶1（PAK1）（效应）和GFP-TrioD1（全球环境基金）。 </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>

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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><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>88821</td><td></td></tr><tr><td>Polyethylenimine, branched, average M&lt;sub&gt;w&lt;/sub&gt; ~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&#039;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&amp;gamma;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>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>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.

这个协议设计为计算Rac1的结合伴侣的相对亲和力，而不需要知道的竞争者的精确浓度（ 图1）。蛋白浓度的测定将错误引入并考虑分子之间竞争时在一个信号通路是不需要的。但是，重要的是要知道，两个竞争者具有相同摩尔浓度的储备溶液，以允许简单的比率来计算的添加不同量的测定时。 40微升的GFP-陷阱珠具有的〜300皮摩尔如此汇合75厘米2烧瓶高度表达细胞将饱和的珠子，其结果是在两个不同的结合蛋白的制剂将是调整前相似的结合能力（图2A）。如果蛋白质之一表示较差，这个问题可以通过纯化蛋白质从细胞中的一个以上的烧瓶被克服。 8（图2B）进行检测。 GEFS优先结合到核苷酸的无GTP酶稳定过渡状态。由于全环基金是丰度低，通常不活跃的，经常涂抹不好，最好是过表达或全球环境基金GEF片段的核苷酸检测无GTP酶。我们经常使用第一张双人同源重奏的，表示为GFP融合（GFP-TrioD1 9）（图2B），但任何环基金会工作。结合对GDP加载GTP酶蛋白质是罕见的。我们最近报道RCC2作为一个这样的蛋白7，占GDP加载可以简单地为宾迪验证纳克既不全球环境基金，也不效应。 从实验的输出将是一个Western印迹描绘绑定到GTP酶两个GFP标记的结合伴侣。通过使用单一抗体来检测两种蛋白质，在该类似量既是竞争对手的结合浓度可被确定，因此，相对亲和力推断。在Rac1的拐蛋白的螺旋桨结构域之间的这种实施例的竞争，coronin-1C（Rac1的结合蛋白A）中，和Rac1的-截存蛋白，RCC2（Rac1的结合蛋白B）证明（图3A）。通过使用coronin-1C螺旋桨（5微升）的恒定体积，并加入RCC2体积增加，我们可以从GFP看到涂抹达到平衡在1.25-2.5微升RCC2（星号）的，这表明RCC2具有更强对于Rac1的比coronin-1C亲和力。通过测量用定量免疫印迹条带的强度，并绘制平均值为每个competitoR，平衡点可以准确地通过识别在该曲线相交（图3B）的卷来计算。 其中一个可能的障碍一个成功的竞争测定法是，如果结合伴侣结合到彼此以及结合Rac1的。在图3A + B我们证明RCC2和coronin-1C的螺旋桨域之间的竞争，而不是全长coronin-1C。之所以使用截短coronin是coronin-1C还通过尾域结合RCC2。当全长coronin-1C滴定针对RCC2，两种蛋白的结合进行检测，由于三元复合物形成，而不是竞争（图3C）。如果竞争的现象发生，一种蛋白质结合会增加，而其他的减少，总的约束GFP融合将保持不变。的情况下一个三元复合物形成，有必要截断GTP酶结合蛋白之一，使得competitors不再进行交互。 <img alt="图1" src="/files/ftp_upload/53254/53254fig1.jpg" /> 图1.工作流程。工作流程来确定使用竞争性试验GTP酶结合蛋白的亲和力的示意图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53254/53254fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53254/53254fig2.jpg" /> 图2.验证纯化的蛋白质。 （A）纯化 GFP标记的Rac1结合通过蛋白质印迹分析的蛋白质，具有抗GFP探测，以确定两个蛋白的相对产率。这在实验期间类型均衡允许调整两种蛋白质的浓度，以便它们在结合实验匹配。（B）中的GD磷，GTPγS无核苷酸加载GST-Rac1的培养与HEK293T裂解表达GFP-TrioD1和约束绘制了内源性PAK1或过表达GFP-TrioD1检测蛋白质。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53254/53254fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53254/53254fig3.jpg" /> 相对蛋白质的图3.蛋白质印迹分析 ，从竞争结合测定结合。实施例输出。（A）中的GDP加载的Rac1中加入5微升的GFP-coronin-1C螺旋桨域和增加的GFP-RCC2的体积滴定，通过Western印迹结合的蛋白质的GFP，与差分检测两种蛋白质的问题被避免和GFP信号报告两个融合蛋白之间的摩尔比。星号表示在eithe竞争比例平衡点的R侧。（B）的来自三个独立实验结合GFP融合蛋白的谱带强度通过定量蛋白质印迹法测定，使用荧光标记的二抗和平均数作图来计算RCC2的量需要以达到平衡。（℃从一个实验，其中Rac1的结合蛋白结合到彼此并形成三元复合物，而不是竞争）实施例输出。 GDP-加载 ​​的Rac1中加入5微升的GFP-RCC2和增加的GFP-coronin-1C全长的体积滴定。在结合的GFP-coronin-1C的增加没有约束的GFP-RCC2的损耗表示三元复合物的形成。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53254/53254fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</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.

比较GTP酶结合蛋白的使用竞争性试验的亲和

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

Universidad San Sebastian

Research

JoVE Journal

Biology

9.1K 次观看.  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.

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

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

科研

生物化学

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

通过共沉淀测量蛋白与F-肌动蛋白的结合

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC的GTP酶激活法

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

生物学

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

亲和沉淀主动RHO上皮细胞裂解液中使用的GST标签的突变Rho蛋白（GST-RhoA蛋白（G17A））全环基金

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

比较GTP酶结合蛋白的使用竞争性试验的亲和

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通过放射性标记的GTP结合测量G蛋白偶联的受体信号

通过共沉淀测量蛋白与F-肌动蛋白的结合

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

RhoC的GTP酶激活法

亲和沉淀主动RHO上皮细胞裂解液中使用的GST标签的突变Rho蛋白（GST-RhoA蛋白（G17A））全环基金

G蛋白偶联受体的基于荧光的钙动员含量表征

蛋白质 - 配体相互作用采用差示扫描荧光法测定

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

比较GTP酶结合蛋白的使用竞争性试验的亲和

摘要

探索更多视频

此视频中的章节

通过放射性标记的GTP结合测量G蛋白偶联的受体信号

通过共沉淀测量蛋白与F-肌动蛋白的结合

膜分馏和 gtpase-关联免疫吸附法检测小 gtpase 预基化和 gtp 结合

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

RhoC的GTP酶激活法

亲和沉淀主动RHO上皮细胞裂解液中使用的GST标签的突变Rho蛋白（GST-RhoA蛋白（G17A））全环基金

G蛋白偶联受体的基于荧光的钙动员含量表征

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

一种基于流式细胞仪的检测方法, 用于识别破坏荧光标记的 CXC 趋化因子配体12到 CXC 趋性因子受体4的化合物

蛋白质 - 配体相互作用采用差示扫描荧光法测定