This work was supported by a grant from the NIH. The assays shown in Figure 1 was performed by Dr. Joseph Fasolo. We thank Dr. Rui Chen for helpful comments.

1.探针制备<ol><li>文化与净化用来检查与其他蛋白质的相互作用如下V5融合激酶探头： <ol><li>用新鲜挑染酵母菌株Y258（的MATa pep4-3，his4-580，ura3-53，leu2-3,112）含V5融合蛋白（从GATEWAY载体pYES-DEST52表示）。使用分离的蛋白质作为探针上的微阵列。板上完全合成 - 尿嘧啶（SC-URA）/ 2％葡萄糖/琼脂酵母和生长在30℃下从冷冻培养（-80℃的甘油原液）3天。 </li><li>从单个菌落接种起子培养（5-20​​毫升）和上摇动平台（220-250转）或车轮在30℃在钪浦/ 2％葡萄糖生长过夜。 </li><li>第二天早晨，接种将400ml钪浦/ 2％棉子糖与足够起子培养培养物至最终OD 600为0.1。 </li><li>成长到接种OD 600为0.6接着半乳糖诱导V5激酶福锡永构建表达通过添加3×酵母提取物/蛋白胨（YEP）补充有6％的半乳糖的溶液中，通过添加足够的由3倍稀释的诱导培养基，以便半乳糖的那个最终浓度为2％。 </li><li>诱导细胞在30℃下进行6小时的摇动平台上。使用2升锥形瓶中，以保证适当的通风。 </li><li>使用JA-10（或可比）转子通过纺丝400毫升细胞悬浮液在1000×g离心5分钟，在4℃下收获细胞。 </li><li>用50ml冰冷的PBS缓冲液并转移至50ml锥形管一次洗细胞。在冰冷的PBS缓冲液再次洗涤沉淀（没有清洁剂或其它添加剂），用于裂解和转移至2ml单元帽管裂解。 </li><li>在4℃旋转细胞在20,000×g离心1分钟以粒料和吸管离开缓冲区。 </li><li>管放置在冰上进行裂解步骤。 </li><li>裂解细胞（250-350微升粒料）用01：在1.5 1mm的氧化锆珠粒1体积细胞沉淀，珠子，和磷酸盐缓冲盐水（PBS）的裂解缓冲液，涡旋使用搅动平台3次将混合物在2分钟的间隔在4℃。 </li><li>离心裂解物在20,000×g离心在台式微量离心机，在4℃下10分钟。 </li><li>吸管上清液进聚碳酸酯高速离心管中并通过ulracentrufugation以150,000×g离心澄清30分钟，在4℃。 </li><li>转移的澄清的裂解物，以含有预洗涤的Ni 2+亲和树脂（〜100微升）的管和孵育在章动器2小时，在4℃下以捕获组氨酸（His）的6X标记V5-融合蛋白。 </li><li>为10分钟，在4℃下用洗涤缓冲液洗树脂三次。沉淀用台式离心机5分钟的树脂在1000 xg离心在4℃下并吸出上清液。加入新鲜的洗涤缓冲液，每次洗涤和洗涤过程中返回管中的章动器的搅拌。 </li><li>洗净后，STEps的是完整的，应用该洗涤树脂至新的G-25柱洗脱。 </li><li>申请25微升洗脱缓冲液中逐步的方式开始用100mM咪唑到500mM咪唑的50mM增量为总共9馏分。 </li><li>利用十二烷基硫酸钠聚丙烯酰胺凝胶测定所收集的级分（SDS-PAGE）和考马斯染色来确定最高度富集的馏分（秒）测定中使用，并添加甘油至30％，并储存于-80℃直至在使用检测。 </li></ol></li></ol> 2.在探测阵列注：请参阅本节的步骤2.6开始试验前的准备抗体溶液。 <ol><li>为了检测的相互作用，稀释V5-荧光团缀合的抗体（ 即 AlexaFluor 647），以在探针缓冲器260纳克/毫升和振荡调匀。 注：准备该抗体溶液30分钟，在使用前和将管上的nutat或者或轮，以确保完全混合和抗体的均匀悬浮液。 </li><li>稀释V5-融合蛋白探针，5-500微克/ ml的浓度范围。 注：优化每个蛋白质 - 蛋白质相互作用测定法中，使用探针的缓冲区。优化涉及增加更多的探针探测缓冲区。通常为10微克/毫升作为起点，并相应地根据信号强度调整。 </li><li>从冰箱中（-20℃）除去蛋白质芯片和带至4℃的冰箱在使用之前。 </li><li>直接添加封闭缓冲液到含有蛋白质micorarrays载玻片支架和盖顶用石蜡膜，防止泄漏。通过在50rpm在舞台上，在4℃振荡方框在封闭缓冲液中进行1小时的阵列。 </li><li>封闭后，将阵列转移到湿润的腔室冷却至4℃，并加入90微升稀释的探针直接到阵列表面。叠加的ARR带有凸起挺杆滑AYS孵育静态（无晃动）在加湿室中在4℃下1.5小时。 </li><li>在探针缓冲器在三个50ml锥形管中的每个1分钟洗阵列3次。滑动添加到含有足够预冷探针缓冲器完全包住滑动锥形管。允许升降滑轻轻滑落的蛋白质微阵列（不要强迫它关闭，因为这可能会导致损坏阵列表面）。 </li><li>作为前完成清洗（步骤2.5），并覆盖有凸起的防滑起苗后立即申请直接抗体溶液阵列。在加湿室中孵育阵列30分钟，在4℃。 </li><li>在台式离心机5分钟在室温下以800×g离心执行相同的洗涤步骤之前（3次1在探针缓冲min），而旋在50ml锥形管中。风干在滑动保持器阵列在黑暗中以扫描该阵列在647毫微米之前30分钟。 </li></ol>

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The authors declare that they have no competing financial interests.

提出最初执行使用85独特酵母蛋白激酶-V5融合蛋白进行比较跨越导致新激酶相互作用网络体内 1的识别酵母蛋白激酶不同及相关家族结合活性的协议。作为一个新兴的蛋白质组学分析技术，高通量（HTP）使用蛋白质微阵列依靠肽库，和真核和原核模式生物蛋白质组8,17筛选的发展;后来，这个实验平台应用于高等真核生物如植物和人类5,7。目前还没有商业上可得到的酵?...

执行蛋白质生物化学和结合的体内活性的全局分析的需要已导致对分析蛋白质-蛋白质相互作用（质子泵抑制剂）和整个蛋白质组1,3-8的翻译后修饰的新方法的发展。蛋白质微阵列被制造为包含抗体10,11-功能蛋白微阵列使用全长功能蛋白4-6,8,9，或分析蛋白质微阵列。它们被加工成含有高密度排列到显微镜载玻片用各种表面化学的蛋白，以促进所需的多种用于进行广泛的生化分析12的实验条件。硝化纤维素和醛的表面化学进行化学附着，通过赖氨酸或亲和力连接方法如镍螯合载玻片用于附His标签的蛋白质，谷胱甘肽对除其他13亲和力附件。 </p&gt; 使用功能的蛋白质微阵列来检测蛋白质-蛋白质相互作用，需要获得一个高质量的功能性蛋白质库14。S.酵母是适合于通过高拷贝亲和力配对产生这样的库标记蛋白构建与高通量色谱纯化技术。酵母基因组的绝大部分已测序，几乎整个蛋白质组可以从高拷贝质粒纯化被表达和生化分析12。一旦蛋白被获得并排列在384孔格式，它们被打印到载玻片允许快速并行多参数生化分析和生物信息学的询问8,14-16。蛋白质微阵列已经用于酶测定和相互作用与蛋白质，脂质，小分子和核酸中的许多其它应用。蛋白质的蛋白质组的表面上的辅助阵列使得它们适合于不同类型的分析检测的，包括免疫亲和，表面等离子体共振，荧光技术及其他技术。此外，它允许精细控制的实验条件下在那里它可能是很难做到的体内 。 该协议的目的是要证明的适当使用的功能性蛋白微阵列来检测蛋白质 - 蛋白质相互作用。此应用程序的使用的兴趣高度纯化的分析物（蛋白质）的蛋白质结合活性的高通量平行生化分析。 C-末端（羧基末端）标记的感兴趣的V5融合诱饵蛋白从在酵母菌株的蛋白质纯化优化的高拷贝质粒制备。 C-末端标记确保了全长蛋白质被翻译。在这项研究中使用的蛋白质是Tda1-V5融合蛋白激酶，其使用镍亲和树脂经由His6X标签纯化。该Tda1-V5融合construct为通过串行洗脱使用咪唑梯​​度洗脱最高度富集的馏分在测定法中使用纯化。

<table border="0" cellpadding="0" cellspacing="0" width="855">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Petri Dishes</td>
			<td style="width:180px;">VWR</td>
			<td>NC-10747</td>
			<td>or comparable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto yeast extract</td>
			<td style="width:180px;">Difco</td>
			<td>0217-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto peptone</td>
			<td style="width:180px;">Difco</td>
			<td>0118-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:180px;">Difco</td>
			<td>0140-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dextrose</td>
			<td style="width:180px;">Sigma</td>
			<td>D9434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Raffinose</td>
			<td style="width:180px;">Sigma</td>
			<td>R0514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Galactose</td>
			<td style="width:180px;">Sigma</td>
			<td>G0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sc-Ura drop out media</td>
			<td style="width:180px;">MP Bio</td>
			<td>114410622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Small Culture tubes</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Large Erlenmeyer Flask</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge JA-10 rotor</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge tubes (500 mL)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Falcon tube (50 mL)</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td>21008-951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PBS Tablets</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Tubes(2ml screw cap tube)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FastPrep Machine</td>
			<td style="width:180px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">116004500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Zircon Beads</td>
			<td style="width:180px;">BioSpec</td>
			<td align="right" style="width:119px;">11079105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Triton X100</td>
			<td style="width:180px;">Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DTT</td>
			<td style="width:180px;">Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">MgCl2</td>
			<td style="width:180px;">Sigma</td>
			<td>M8266&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">NaCl</td>
			<td style="width:180px;">Sigma</td>
			<td>S3014&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Imidazole (pH = 7.4)</td>
			<td style="width:180px;">Sigma</td>
			<td>I5513&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">PMSF</td>
			<td style="width:180px;">Sigma</td>
			<td>93482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Complete Inhibitor Cocktail (EDTA Free)</td>
			<td style="width:180px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Phosphatase inhibitor cocktail 1</td>
			<td style="width:180px;">sigma</td>
			<td>P2850</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BSA</td>
			<td style="width:180px;">Sigma</td>
			<td>A2153&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Tween-20</td>
			<td style="width:180px;">Sigma</td>
			<td>P1379&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">ATP</td>
			<td style="width:180px;">Sigma</td>
			<td>A1852&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Shaking Incubator (30 &#176;C)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nickel Affinity Resin</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R901-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">G25 column</td>
			<td style="width:180px;">GE life sciences</td>
			<td>27-5325-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Nutator</td>
			<td style="width:180px;">VWR/Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">SDS-PAGE Gel (NuPage)</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Coomassie Blue Stain</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>R960-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Alexa647 Tagged monoclonal V5 Antibody</td>
			<td style="width:180px;">Life Technologies</td>
			<td>451098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Protein Microarrays Kit</td>
			<td style="width:180px;">Life Technologies</td>
			<td>PAH0525013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Genepix Scanner</td>
			<td style="width:180px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:267px;">Lifter Slips</td>
			<td style="width:180px;">Thomas Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:291px;">http://www.thomassci.com/Supplies/Microscope-Cover-Glass/_/LIFTER-SLIPS/</td>
		</tr>
		<tr height="124">
			<td height="124" style="height:124px;width:267px;">Lysis Buffer: 0.1% Triton X-100, 0.5 mM DTT, 
			2 mM MgCl2, 500 mM NaCl, 50 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF&#160; (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride; pH 7.4) to obtain the desired volume of lysis solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">Blocking Buffer: 1% bovine serum albumin (BSA), 0.1% Tween-20</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagents to 1 X PBS to obtain the desired volume of blocking buffer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:267px;">Probe Buffer: 2 mM MgCl2, 0.5 mM DTT, 0.05% Triton X-100, 50 mM NaCl,500 &#956;M ATP (for kinases), 1% BSA</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:267px;">Wash Buffer: 0.1% Triton X-100, 500 mM NaCl, 0.5 mM DTT, 50 mM imidazole (pH 7.4),2 mM MgCl2</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:267px;">Elution Buffer: 0.1% Triton X-100, 0.5 mM DTT, 2 mM MgCl2, 500 mM NaCl, 50 &#8211; 500 mM imidazole (pH 7.4), Complete protease inhibitor cocktail &#8211; EDTA-free, Phosphatase inhibitor Cocktail 1, 1 mM PMSF (add just prior to use)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:291px;">Before you begin, it is important to note the concentration gradient of imidazole (50 &#8211; 500 mM) and to create separate tubes for each concentration (it is advisable to create the interval using 50 mM increments). Add the reagent to 1 X PBS to obtain desired volume of probe buffer</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:267px;">3x YEP +6% galactose: Dissolve 30 g of yeast extract and&#160; 60 g of peptone in 700 ml of H2O, and autoclave. Add 300 ml of filter sterilized 20% galactose (wt/vol)</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;galactose should not be autoclaved</td>
		</tr>
	</tbody>
</table>

probing high-density functional protein microarrays to detect protein-protein interactions

含〜4200重组酵母蛋白的高密度功能蛋白微阵列检查使用含有用于读出一个V5表位标签的亲和纯化的酵母激酶融合蛋白激酶蛋白质 - 蛋白质相互作用。纯化激酶通过酵母菌株的高仿蛋白质生产带有含下GAL诱导启动子激酶-V5融合构建的质粒优化培养获得。酵母生长在限制性培养基用中性碳源为6小时，随后通过感应用2％半乳糖。接着，将培养物收获和激酶用标准亲和层析技术来获得用于在测定中使用高度纯化的蛋白激酶纯化。将纯化的激酶稀释激酶缓冲到一个适当的范围内用于测定和蛋白微阵列被阻断之前与蛋白质微阵列杂交。该杂交后，阵列探测用单克隆V5 antibODY鉴定蛋白质结合的由激酶-V5蛋白。最后，该阵列是利用标准的微阵列扫描仪扫描，并且数据被提取用于下游信息学分析1,2，以确定高置信度设置蛋白质相互作用的体内下游验证。

使用标准的芯片读取器，以评估Tda1-V5蛋白激酶融合构建体对含有约4200独特S.酵母功能性蛋白微阵列诱饵蛋白质的蛋白质-蛋白质相互作用的活性，观察酵母 GST-融合蛋白。与Genepix软件进一步审讯发现多种结合不同强度的活动。的亲和力从单克隆V5-荧光团缀合的抗体结合至其靶（V5-激酶）导出的信号的梯度强度衡量。 ProCat评分算法2用于鉴定蛋白质-蛋白质相互作用跨两个单独的...

Watch this Scientific Journal Video about Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions at JoVE.com

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

使用含有几乎整个S.蛋白质芯片酵母蛋白质组被探查以十万计的蛋白质-蛋白质相互作用中并行快速无偏询问。这种方法可以用于蛋白质 - 小分子，翻译后修饰，并在高通量其它测定。

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

probing-high-density-functional-protein-microarrays-to-detect-protein

Research

JoVE Journal

Biology

7.9K 次观看.  Stanford University. 使用含有几乎整个S.蛋白质芯片酵母蛋白质组被探查以十万计的蛋白质-蛋白质相互作用中并行快速无偏询问。这种方法可以用于蛋白质 - 小分子，翻译后修饰，并在高通量其它测定。 

视频: Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

科研

生物学

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

鉴定蛋白质相互作用的网站使用多肽阵列

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换：应用程序的设计和开发非ATP竞争性CDK抑制剂

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...