VN是由英国皇家学会的支持。 JPR是澳大利亚研究理事会未来研究员（FT0992129）。我们感谢仪吉福德博士批判地阅读手稿。

注：所有的步骤都在室温下进行，除非另有说明。 1。植物材料和缓冲器的制备<ol><li> 对于N。本生 <ol><li>生长的根癌土壤杆菌的菌株息进行瞬时表达（在此示例PRF-FLAG，PRF-3xHA，PTO-FLAG和空载体作为对照）通过在28在液体培养基（含有适当抗生素的L培养基）摇动（200rpm）下°C，直到固定相。 </li><li>收集和沉淀农杆菌通过离心（3,000×g离心5分钟）。弃上清，重悬制粒农杆菌中渗透的缓冲。 </li><li>通过获得的光密度（OD）值在600nm的吸光率（Abs 600纳米）测量土壤杆菌的量。调整细菌的OD到0.1-0.8。 </li><li>渗透4周老N。植物本生 （22℃，16小时光照）与农杆菌由汉D（用1毫升无针头的注射器），或通过真空（加0.02％体积/体积表面活性剂）。 注意：使用第一近乎完全展开的叶片，两个紧接在老叶为最有效​​的蛋白表达。对于检测泛素化或乙酰化蛋白的渗透叶子用100nM的MG-132或100毫微克/毫升曲古抑菌素A，分别在1和2天后浸润。 </li><li>收获叶浸润2-5天后渗透和冷冻在液氮中。存储样本中的-80℃冰箱。 注：事先通过计算样本超过5天的时间过程，并通过免疫印迹法检测蛋白水平确定相关蛋白表达的最好水平。 </li></ol></li><li> 对于A。稳定拟南芥转基因株系 <ol><li> A.成长拟南芥种子在6孔板中补充用5ml MS液体培养基（1％w / v的蔗糖）中，每孔。放三到转基因一行五种子（消毒和分层）利息或每孔未转化的对照线。摇以200rpm板转移到生长室的前两天，离开2周（22℃，10小时光照）。 </li><li>收获样品并冷冻在液氮中。 注意：您可以作为控制用的表达无关蛋白具有相同标签的目的蛋白的转基因系。 </li></ol></li><li> 缓冲器 <ol><li>准备缓冲A.德气体缓冲1-2小时（对于类受体蛋白激酶，其他膜结合蛋白和核蛋白，加1％V / V IGEPAL CA-630 53）。 注意：你可以保持缓冲液A在4℃下去。你可以用1％V / V的Triton代替IGEPAL CA-360。 </li><li>提取准备前加入80ml冷的缓冲液B至少1-2小时。 注意：组织与提取缓冲液的理想的比例为1:4（重量/体积）。 </li></ol></li></ol> 2。蛋白抽提<ol><li>之前，为了提取准备加入80ml冷的缓冲液C。 <b转/&gt;注：添加10μMMG-132对泛素化蛋白的检测。 </li><li>磨20克N的本塞姆氏组织（约15叶）或A。用研钵和研杵拟南芥幼苗（每6孔板2-4克）在液氮中。 </li><li>加上80毫升冷的缓冲液C，以各20 g地组织，拌匀，和解冻的冰。 注：研所有样品的同时（利息及控制蛋白质）。 </li><li>均质与3连发每个10秒的全速组织匀浆器（预冷4℃）。通过在4 22-25微米的组织和离心机以30,000×g离心30分钟过滤 °C。 注：此外，同质化的新鲜预冷的研钵和杵。 </li><li>对于阻塞和洗涤步骤的亲和基质，配制20 ml缓冲液D的</li><li>用500ml冷缓冲液D中含1％BSA为5分钟，在4块100毫升合适的亲和基质 °C。 注意：首选AF菲妮迪矩阵包括抗FLAG M2琼脂糖，链亲和素琼脂糖，GFP-Trap_A和抗HA琼脂糖。 </li><li>洗3次用1毫升冷缓冲液D。 </li><li>通过0.45μm的注射器过滤器除去叶提取物和过滤器的上清液放入冰上的一个新的试管中。 注：该提取物的颜色应为绿色或黄色;如果样品已经变成褐色，放弃并重新开始。 </li><li>取粗提物进行免疫印迹的等分试样，加入5×SDS-PAGE上样缓冲液，涡旋，并通过煮沸10分钟的样品在80-100变性 °C下加热块。 </li></ol> 3。蛋白免疫沉淀<ol><li>分裂的蛋白质提取物分成两个50ml试管并加入50微升的亲和基质悬浮在缓冲液D以每管。用2小时轻柔旋转孵育在4°C 注：长时间孵育也没有发现增加产量。 </li><li>通过增加编制600微升的洗脱缓冲液（亲和基质的6倍珠体积）缓冲液D（终浓度）：0.5％BSA，0.25 mg / ml的FLAG肽（抗-FLAG M2琼脂糖）或10mM的D-生物素（为链亲和素琼脂糖）。 注：洗脱不建议在GFP-Trap_A和抗HA矩阵的情况下，所以直接进行在1x SDS-PAGE上样缓冲液中煮沸，步骤3.11。 </li><li>通过离心分离沉淀的亲和基质，在4℃（5分钟，在1,000×g离心）。 </li><li>取未结合的提取物进行免疫印迹的等分试样，弃去上清液的其余部分，留下约500μl的在管的底部。 </li><li>重悬带宽口端部的浆料溶液。 </li><li>传输从两个管将混合浆料溶液到单个1.5毫升管。 注意：从现在起，使用绑定离心管1.5毫升低蛋白。 </li><li>脉冲3次，持续5秒使用台式离心机，以沉淀出的亲和基质，去除上清。 </li><li>洗3到5倍，用1ml冷的缓冲液D的洗涤沉淀之间的亲和力马特里x先前所描述的并丢弃上清液。在最后一次洗涤除去过量的缓冲液D，用注射器的针头。 注意：在使用缓冲液D用0.1％IGEPAL甲最终洗涤可用于进一步减少非特异性结合。 </li><li>用200微升适当的洗脱缓冲液（抗-FLAG M2或链亲和素琼脂糖见步骤3.2）3次，每次5 min洗脱持续振荡培养。 </li><li>汇集3洗脱物在单管中。用30微升吸附树脂的浓缩蛋白质。涡，静置5分钟，沉淀出树脂（以10,000×g离心2分钟）。弃上清。 注意：对于GFP-Trap_A和抗HA琼脂糖，跳过步骤3.9和3.10。 </li><li>加50微升1X SDS-PAGE上样缓冲液中，以沉淀出的亲和度矩阵或吸收树脂。涡和熬在80-100 10分钟 °C下加热块。 </li><li>离心煮沸亲和基质或树脂为2分钟，在10,000×g下。分装5微升的supernatan的t的免疫印迹，并用其它馏分运行到〜10厘米长的SDS-PAGE凝胶（ 图2）。 </li><li>用于分离和鉴定磷酸化蛋白的质谱，装入剩余的45微升到〜19厘米长的SDS-PAGE凝胶，如果额外的分离是必要的（ 图2）。 注意：空白车道可以包含所有的样品之间，以减少样品的污染。 </li></ol> 4。蛋白质的消化<ol><li>染色用胶体考马斯亮蓝（CCBB）染色的SDS-PAGE凝胶。 注：最小化处理的凝胶，以减少污染与角质。确保没有装备或工具已被用于免疫印迹或可能已经离开残余蛋白污染物的其它活动。 </li><li>脱色用大量水洗，最好的O / N的凝胶从用干净的刀片凝胶中切感兴趣的频带。 注：对准免疫印迹凝胶如果necessaRY找到正确的区域。作为磷酸化可以延迟蛋白质的迁移在SDS-PAGE紧接的上方和下方所关心的频带切的区域。 </li><li>切块的凝胶切片成2-4毫米的立方体（这确保了凝胶将不阻塞移液器吸头被覆盖在管的解决方案）。放置在一个试管中。 </li><li>估计保持良好淹没凝胶所需的音量。使用此金额为衍生，孵化与蛋白酶和肽提取，然后用双三重卷洗。 注意：在一个典型的具有大约100μl的切割凝胶条的消化，用于洗涤500μl的溶液，2×180微升进行脱水，加入150μl的减少，120μl的用于烷基化，和120微升消化。 </li><li>通过加入50％乙腈（在50mM碳酸氢铵中，ABC）洗凝胶块2×20分钟（或直到脱色）。移液器起飞时注意不要删除凝胶块的解决方案。 </li><li>脱水用100％乙酰腈5分钟，除去游离的液体。 注：凝胶应该出现萎缩和白色。如果蓝色仍然存在，孵育时间在乙腈/ ABC和/或在升高的温度下（45-55℃）。 </li><li>减少蛋白质二硫键，加入10 mM的DTT在水中孵育30-45分钟，在56ºC下振荡。除去游离液体。 </li><li>烷基化的半胱氨酸残基通过加入55 mM的氯乙酰胺（在50mM ABC）20-30分钟（室温，避光）。除去游离液体。 </li><li>用50％乙腈（在50mM ABC）洗凝胶块2×10分钟。除去游离液体。 </li><li>脱水凝胶片用100％乙腈5分钟，除去游离的液体。 </li><li>对于胰蛋白酶消化，加入40微升的胰蛋白酶工作液（在消化缓冲液100毫微克胰蛋白酶：50毫米农行，5％乙腈，平均考马斯亮蓝染色带）。允许凝胶片再水合10分钟。添加的消化缓冲不够的，如果需要覆盖凝胶块。在37ºC的O / N。 </li><li>停止消化，并从凝胶块中提取的肽，添加5％的甲酸（50％乙腈）（加相同体积的消化量）。超声处理5-10分钟。将上清转移至新管。重复提取3倍。 </li><li>用冷冻干燥法（首选）或真空浓缩器（如速度，Vac或萨文特）干燥肽。储存于-20ºC。 </li><li>提交您的样品进行质谱分析。 注：我们提交我们的样品在塞恩斯伯里实验室（英国诺里奇）质谱设施。协议在质谱设施有很大的不同，但通常将肽溶解于0.2％三氟乙酸中含有2％乙腈装载到耦合到电喷雾串联质谱法液相色谱系统之前。 </li></ol>

作者宣称，他们有没有竞争的利益（财务或其他）。

通过的PAMP和效应阐明受体激活的机制，可以极大地促进我们的植物免疫力的理解作出贡献。在过去的20年中，基因和酵母双杂交筛选是有助的蓝耳病和NB-LRR蛋白的发现。最近，基于质谱的协议已经建立了蛋白质的免疫信号55-58，其翻译后修饰11,33,59,60中差异调节的鉴定，免疫复合物61和效应的组合物的目标62。这里，我们描述一个简单的协议，用于翻译后修饰调节免疫复合物的活化的鉴定。 与前面所述的协议相比，该协议允许的翻译后修饰的动态变化的详细鉴定。大规模的蛋白质组方法的协议可以识别蛋白质的翻译后修饰，但不能够揭示由于限制了翻译后修饰的位点可塑性编辑的蛋白质。在蛋白质磷酸化的情况下，大型蛋白质组学方法通常只能识别的主要磷酸化位点11,33,59。的蛋白的磷酸化状态的详细的表征需要的蛋白质，可以仅通过目标蛋白47的部分纯化获得大量。在这里描述的协议伴侣的蛋白纯化方法，得到了目的蛋白的大量，用纯化的蛋白质的质谱分析。根据这一协议山口的单磷酸化事件主要归因于丝氨酸198，如前所述52，但有些质谱谱图也支持苏-195或Thr-199单磷酸化事件。当观察到的PTO双磷酸化，磷酸化氨基酸的主要组合为丝氨酸198和Thr-199虽然也观察到在其他网站组合（表1）。这些结果清楚地表明蛋白激酶的磷酸化位点的可塑性和所描述的协议，以在细节所有可能的磷酸化位点的特征的能力。 该协议中最关键的步骤是：蛋白质1）足够的提取; 2）保护翻译后修饰的，和靶蛋白3）足够量。首先，对蛋白质的充分萃取，研磨的组织在液氮中，并随后在协议中所描述的是使用组织匀浆器是很重要的。如果组织匀浆器不可用时，用研钵和研杵都可以使用。同样重要的是要使用一个的比率，以三（或四）的克组织的提取缓冲液体积。这个组织以缓冲和比率，我们建议的高强度缓冲液将确保该pH值将在提取过程中保持中立。我们发现，这是特别重要的为A。拟南芥和番茄额外ctions 52。保护翻译后修饰可通过包括在所有的缓冲区酶，可以除去翻译后修饰的合适的抑制剂来实现。同样重要的是要执行的所有步骤在4℃和预冷的缓冲液和仪器。靶蛋白的产量更高，可以通过使用植物表达该蛋白质以足够的量，并通过使用高含量的组织（约20g）来实现。为获得足够数量的靶蛋白的最关键的一步是通过直接免疫沉淀浓缩靶蛋白。这一步的意义是由我们失败的Prf免疫沉淀后，以确定的PTO翻译后修饰，由于共沉淀PTO（ 图2B），数量有限高亮显示。与此相反，的PTO直接免疫沉淀，得到实质上更多可测量的肽（ 图2C），导致约80％的覆盖率的蛋白质序列和翻译后修饰鉴定（ 表1）中的。我们还STrongly推荐使用表位标记的蛋白免疫沉淀。在比较了针对天然蛋白表位标记亲和基质抗体可产生更高的金额部分纯化的蛋白质。使用针对的免疫原生蛋白质的Pto 51（ 图2A）的抗体，我们能够确定的PTO两个主要的磷酸化位点只有单一的磷酸化。 这个协议主要被开发用于N的部分纯化本生胞浆蛋白和随后的鉴定及其磷酸化位点， 但使用本文描述的修改，该协议可以很容易地适应A。拟南芥蛋白和膜结合蛋白。这个协议的主要目的是，得到足够量的靶蛋白的翻译后修饰进行的识别，而不是实现复杂的最高纯度。如果需要更高纯度是必需的复杂的提纯的第二步骤可以被添加到这个协议52。在这种情况下，第一步骤将允许从无需使用高盐或酸的和随后的步骤中的亲和基质洗脱目标蛋白，就可能需要一个矩阵，它要求苛刻的洗脱条件。需要强调的是，我们只测试了这个协议的磷酸化位点的鉴定和我们目前正在测试其能力的额外翻译后修饰细节识别是很重要的。 我们的代表结果清楚地表明蛋白激酶的磷酸化位点的可塑性和所描述的协议来表征磷酸化位点的能力。最重要的是，他们强调的是识别磷酸化位点通过质谱法和通过自动磷酸化位点的单个点突变依赖于蛋白质的低量的可给予混淆的结果。我们在这里展示和先前47 </sup&gt;的PTO上丝氨酸-198和Thr-199是双磷酸化与PRF /的Pto复合物的活化相关联。相比之下，以前的发布结果47,63表明，丝氨酸198和Thr-199的单点突变为丙氨酸，能防止磷酸化在这些网站上，有能力的信号表明这些网站的磷酸化是不复杂的激活的先决条件。这些结果现在可以通过磷酸化的辅助站点的Thr-195的解释。类似的洞察其他蛋白激酶的磷酸化位点的可塑性可以按照此协议来获得。此外，使用磷酸化位点相结合的办法点突变，蛋白质，可以抑制特定的蛋白激酶（效应物）结合蛋白免疫沉淀和质谱分析将导致一个更好的了解蛋白质激酶的磷酸化位点的可塑性的进化意义。

免疫途径依赖于蛋白质的各种翻译后修饰（翻译后修饰），以迅速地转导信号并激活免疫应答1。翻译后修饰是蛋白质结构的快速，可逆的，高度特异性的化学变化，可以影响蛋白质的构象，活性，稳定性，定位，和蛋白质-蛋白质相互作用2-4。在植物中，超过300种翻译后修饰已经确定，包括泛素化，SUMO化，硫酸化，糖基化和磷酸化2,5。越来越多的证据机身凸显翻译后修饰在植物免疫1,5,6的不同方面的重要性。蛋白质的磷酸化，可逆附着一个磷酸基团的一个丝氨酸，苏氨酸或酪氨酸残基是许多细胞功能的调节，而不是令人惊讶的最高度研究PTM在植物防御信号级联5-11。磷酸化依赖的信号是植物防卫厅的一个组成部分微生物胞外感知由跨膜受体，或细胞内承认多结构域蛋白质阻力后5,8化酶激活启动。 根据入侵植物，微生物是通过质膜受体称为模式识别受体（的PRRs）12进行检测。已知的PRR要么是受体样蛋白（RLPS）或受体样激酶（类受体蛋白激酶），两者都携带一个配体结合胞外域和一个单一的跨膜结构域。在对比RLPS，类受体蛋白激酶具有胞内激酶结构域13-15。模式识别受体的胞外域结合的保守微生物诱发剂分子，称为病原体相关分子模式（PAMP）的领导，在类受体蛋白激酶的情况下，向细胞内结构域6,16内的自身磷酸化和磷酸转移。下游猪蓝耳病感知诱导磷酸化，胞质蛋白激酶包括17以后的磷酸化， </suP&gt; 18，E3连接酶19，有丝分裂原活化蛋白激酶（系）20，21，钙依赖性蛋白激酶（CDPK）22，23，和转录因子24，25导致PAMP触发免疫（PTI）。 除了细胞外感知通过模式识别受体，细胞内识别病原体通过细胞质受体的实现。这些多域的电阻（R）的蛋白质包含一个可变的N-末端结构域，中央核苷酸结合（NB）基序，和C-末端富含亮氨酸的重复序列（LRR），因此被称为NB-LRR蛋白26,27。 R蛋白直接或间接地识别来源于病原体的毒力的分子，称为效应器，也被称为目标的PRR和免疫系统的其他节点。识别引起强烈抗辩，导致效应触发免疫（ETI）28-31。 NB-LRR蛋白具有requisiTE ATP结合基序的NB域30，32内，但他们缺乏一个激酶结构域。 NB-外LRRs的保守结构域的磷酸化已经报道在大规模蛋白质组调查33，但其为ETI相关性还不清楚。同样以公共交通交汇处，激活NB-LRR蛋白导致磷酸化细胞质蛋白包括的MAPK 34-37和38-40 CDPK基因的。最重要的是，效应识别可导致磷酸化的辅助蛋白直接相互作用与NB-LRR蛋白41。一些例子包括RIN4（ 拟南芥 ）和PTO（番茄，茄lycopersicum）与NB-LRR蛋白相互作用的蛋白质，RPM1 42，43和44的Prf分别。在感染野火病菌的效应蛋白AVRB RIN4诱导磷酸化，最有可能的受体类激酶RIPK 45， <sup&gt; 46。同样，在番茄的P。丁香效应AvrPto和AvrPtoB诱导磷酸化的Pto 47。在对比RIN4它缺乏一个激酶结构域，且必须反式磷酸化，PTO处于能够自动磷酸化48和反式磷酸化底物49，50的有效激酶。而PTO要求其对信号的效应依赖的启动激酶活性，激酶死亡突变的Pto仍然能以效应无关的方式51的信号。最近，我们通过证明的PRF /复杂的Pto低聚，包含多个的Prf和PTO分子52，并且在同一个复杂分子的Pto可以跨磷酸对方47解释了这些意见。我们提出了一个模型，其中PTO（传感器）的一个分子与效应蛋白相互作用，引起构象变化到NB-LRR蛋白（PRF），反过来激活第二的Pto分子（帮手）机智欣复杂。随后，辅助分子的Pto反式磷酸化传感器的Pto通向电阻复杂47的完全活化。 这些例子表明，免疫复合部件及其潜在翻译后修饰后效应器识别的识别可导致更好地理解信号如何从效应器感知导至下游目标。在这里，我们描述了NB-LRR型相互作用的蛋白质和随后的鉴定及其翻译后修饰的蛋白质纯化方法。我们用本氏烟草和番茄PRF /复杂的Pto作为一个模型，但是相同的方案可以很容易地被应用到类受体蛋白激酶从N。本生和A.拟南芥 53小的修改，因为我们在协议中说明。

<table border="0" cellpadding="0" cellspacing="0" width="1108">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:296px;">MgCl2</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:181px;">M8266</td>
			<td style="width:436px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES</td>
			<td>Sigma</td>
			<td>M8250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
			<td>toxic - 1 M stock in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 1mL sterile</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe needle</td>
			<td>Terumo</td>
			<td>NN-2525R&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silwet L-77 (surfactant)</td>
			<td>Lehle Seeds</td>
			<td>VIS-01</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MG-132 (Z-Leu-Leu-Leu-al)</td>
			<td>Sigma</td>
			<td>C2211</td>
			<td>1 M stock in DMSO, store at -80 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MS salts</td>
			<td>Sigma</td>
			<td>M5524</td>
			<td>oxidizing, toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>Sigma</td>
			<td>16104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>adjust pH with 1 N HCl to make 1 M Tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>5 M stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td>toxic - 0.5 M stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>National Diagnostics</td>
			<td>EC-606</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGEPAL CA-630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PVPP (polyvinylpolypyrrolidone)</td>
			<td>Sigma</td>
			<td>P5288</td>
			<td>do not confuse with PVP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DTT (DL-dithiothreitol)</td>
			<td>Sigma</td>
			<td>43815</td>
			<td>toxic - 1 M stock, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plant protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P9599</td>
			<td>do not freeze/thaw too many times</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PMSF (phenylmethylsulfonyl fluoride)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td>corrosive, toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calyculin A</td>
			<td>Cell Signaling Technology</td>
			<td>9902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Fluoride (NaF)</td>
			<td>Sigma</td>
			<td>S7920</td>
			<td>toxic - 1 M stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Molybdate (Na2MoO4)</td>
			<td>Sigma</td>
			<td>S6646</td>
			<td>0.5 M stock</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;">Sodium orthovanadate (Na3VO4)</td>
			<td>Sigma</td>
			<td>450243</td>
			<td>toxic - Prepare a 200 mM solution of sodium orthovanadate. Adjust the pH to 10.0 using either 1N NaOH or 1N HCl. The starting pH of the sodium orthovanadate solution may vary with lots of the chemical. At pH 10.0 the solution will be yellow. Boil the solution until it turns colorless (approximately 10 minutes). Cool to room temperature.Readjust the pH to 10.0 and repeat steps 3 and 4 until the solution remains colorless and the pH stabilizes at 10.0.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Okadaic acid</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-3513</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kinematica Polytron tissue homogeniser</td>
			<td>Fisher Scientific</td>
			<td>08-451-320&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Miracloth</td>
			<td>Merck Millipore</td>
			<td>475855-1R&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-FLAG M2 agarose</td>
			<td>Sigma</td>
			<td>A2220</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin-agarose</td>
			<td>Thermo-Scientific</td>
			<td>20347</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GFP-Trap_A</td>
			<td>Chromotek</td>
			<td>gta-20</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-HA-Agarose</td>
			<td>Sigma</td>
			<td>A2095</td>
			<td>this matrix is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.45 &#956;m filter</td>
			<td>VWR</td>
			<td>513-1902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FLAG peptide</td>
			<td>Sigma</td>
			<td>F3290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-biotin</td>
			<td>Sigma</td>
			<td>47868</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">StrataClean resin</td>
			<td>Agilent</td>
			<td>400714</td>
			<td>this absorption resin is recommended for protein precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini-PROTEAN Tetra Cell</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PROTEAN II XL Cell</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SimplyBlue SafeStain</td>
			<td>Invitrogen</td>
			<td>LC6060</td>
			<td>this stain is recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein low-binding tubes (LoBind)</td>
			<td>Eppendorf</td>
			<td>0030 108.116</td>
			<td>these tubes are recommended</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium bicarbonate (ABC)</td>
			<td>Sigma</td>
			<td>9830</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>VWR</td>
			<td>83639</td>
			<td>toxic, flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-chloroacetamide</td>
			<td>Sigma</td>
			<td>22790</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Promega</td>
			<td>V5280</td>
			<td>irritant, sensitizing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma</td>
			<td>14265</td>
			<td>toxic, corrosive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water-bath sonicator</td>
			<td>Ultravawe</td>
			<td>Ultra BT Ultrasonic Bath</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LTQ-Orbitrap XL</td>
			<td>Thermo-Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trichostatin A</td>
			<td>Sigma</td>
			<td>T8552</td>
			<td>toxic</td>
		</tr>
	</tbody>
</table>

identification of post-translational modifications of plant protein complexes

植物迅速适应不断变化的原因阐述感知环境和信号系统。在病原体攻击，植物迅速感染通过免疫复合物的募集和活化反应。免疫复合物激活与翻译后修饰的蛋白质（翻译后修饰），如磷酸化，糖基化，泛素化或关联。了解如何将这些翻译后修饰被编排将导致一个更好地了解如何电阻实现的。 这里我们描述了核苷酸结合富含亮氨酸的重复序列（NB-LRR）相互作用蛋白和随后的鉴定及其翻译后修饰（翻译后修饰）的蛋白质纯化方法。小的修改，该协议可应用于其它植物蛋白质复合物的纯化。该方法是基于对所关注的蛋白，它随后被部分纯化的B的表位标记的版本的表达Ý免疫沉淀，并进行质谱分析鉴定相互作用的蛋白质和翻译后修饰的。 该协议表明：i）条。在翻译后修饰，如磷酸化的动态变化可以通过质谱法进行检测;ⅱ）。有足够数量的感兴趣的蛋白质，这可以补偿缺乏的免疫沉淀物的纯度是很重要的;ⅲ）。为了检测目的蛋白质的翻译后修饰，这种蛋白质具有将免疫沉淀，以获得蛋白质的足够量。

我们在这里描述的协议标签蛋白的纯化稳定的转基因A。拟南芥线或蛋白质的N中瞬时表达后， 本塞姆氏 。 如图1中的靶蛋白的纯化-质谱联用，以允许鉴定相互作用蛋白与靶蛋白的翻译后修饰的。该协议被设计为参与植物免疫和识别它们的翻译后修饰的蛋白质的纯化，但可以应用到任何标记的植物蛋白的纯化。 作为一个例子，我们使用来自N的PRF /复杂的Pto的净化本塞姆氏的转基因N。本塞姆氏线38-12 54，表达35S：PTO，瞬时转化的35S：PRF-FLAG和复杂的免疫沉淀用抗FLAG M2琼脂糖（ 图2A）。在图2A中的免疫印迹（IB）举例说明，大部分的靶蛋白（PRF）的免疫沉淀。 PTO和相互作用的效应蛋白AvrPtoB被共纯化用的Prf和使用抗体和质谱分析（ 图2A和2B）来检测。我们发现，随着共纯化的Prf在PTO迁移较慢的在SDS-PAGE凝胶当与效应子构建体35S共表达：AvrPto和35S：AvrPtoB（ 图2A）。这两种效应蛋白诱导的PRF-交互PTO（ 图2A）的迁移速度较慢。的PTO此效应识别依赖性缓慢迁移先前归因于磷酸化的，因为它可以通过用磷酸酶44处理除去 ​​。为了检测磷酸化位点贡献于PTO的缓慢迁移，我们受到PRF的copurifying到的Pto质谱分析。尽管与高表达的PTO其易于检测与免疫印迹，我们可以检索肽çovering只有57％的Pto序列，我们没有发现任何磷酸化位点（ 图2B）。 随后，我们改变了策略，目标在PTO蛋白与抗FLAG。N。本塞姆氏烟草植物的WT瞬时转化的35S：PRF-3HA和35S：PTO-FLAG，带或不带35S：AvrPto和35S：AvrPtoB的Pto蛋白，其包含两个PRF-络合的和游离的形式，总量为免疫沉淀用抗FLAG M2琼脂糖，并进行SDS-PAGE分级分离，凝胶内胰蛋白酶消化和质谱分析（ 图2C）。用这种方法，我们鉴定的Pto肽跨越其序列的约80％和未知100kDa的相互作用蛋白。我们确定了一套单，双磷酸化位点PTO的肽187-202和188-202（ 表1）。 SER-198和Thr-199是主要的磷酸化位点，但其他河粉还检测到sphorylation位点（ 见表1）。在丝氨酸198和Thr-199最重要的是双磷酸化被认定只有在任一效应蛋白（ 表1）的存在。我们最近发现了类似的一组的Pto磷酸化位点，并示出的双磷酸化事件的意义信令47。下面这里描述的方案，我们能够识别靶蛋白的磷酸化状态的动态变化。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51095/51095fig1highres.jpg" src="/files/ftp_upload/51095/51095fig1.jpg" /> 图1。一般实验设计的靶蛋白进行免疫沉淀（IP）进行标记，并表示在植物中 ，暂时在本氏烟或稳定在拟南芥 。继蛋白质提取靶蛋白免疫沉淀与亲和基质。将蛋白质通过电泳分离的丙烯酰胺凝胶。凝胶带被切除。蛋白质从凝胶中提取的频段，并进行质谱分析。磷酸化位点和相互作用的蛋白质鉴定。 <a href="https://www.jove.com/files/ftp_upload/51095/51095fig1highres.jpg" target="_blank">点击这里查看大图。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51095/51095fig2highres.jpg" src="/files/ftp_upload/51095/51095fig2.jpg" /> 图2。的Prf和PTO免疫A）。构建35S：PRF-FLAG瞬时表达38-12行（35S：PTO）与空载体（EV），35S：AvrPto或35S：AvrPtoB。使用抗FLAG亲和基质三天后浸润的Prf进行免疫沉淀。免疫noblots（IB）进行的Prf，AvrPtoB和PTO被执行。正如通过在免疫印迹的Pto面板底部的箭头所示，AvrPto和AvrPtoB诱导山口B的较慢迁移）。构建35S：PRF-FLAG瞬时表达38-12行（35S：PTO）与空载体（EV），35S：AvrPto或35S：AvrPtoB。使用抗FLAG琼脂糖三天后浸润的Prf免疫沉淀和分离的SDS-PAGE。将凝胶用胶体考马斯亮蓝（CCBB）和可见的Prf，PTO和AvrPtoB蛋白质条带从凝胶切出，并通过质谱法进行分析。质谱鉴定PTO和的Prf序列的百分比肽覆盖被指示。℃）。该构建35S：PRF-3xHA和35S：PTO-FLAG以及35S瞬时表达：AvrPtoB，35S：AvrPto或全空载体（EV） 本塞姆氏 。三天后浸润的总量使用抗FLAG琼脂糖PTO-FLAG免疫沉淀和解决在SDS-PAGE。可见PTO和PTO-相互作用蛋白的条带从凝胶切出，并通过质谱法进行分析。 PTO和100 kDa的PTO-相互作用蛋白带是清晰可见的胶体考马斯亮蓝（CCBB）染色凝胶，而的Prf略少可见的（如箭头所示）。 IP：免疫沉淀; IB：免疫印迹; CBB：考马斯亮蓝染色; CCBB：胶体考马斯亮蓝染色。 <a href="https://www.jove.com/files/ftp_upload/51095/51095fig2highres.jpg" target="_blank">点击这里查看大图。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td colspan="3"> PRF </td></tr><tr><td></td><td rowspan="2">电动车</td><td rowspan="2"> AvrPto </td><td rowspan="2"> AvrPtoB </td></tr><tr><td> PTO肽188-202 </td></tr><tr><td> GTELDQ [P T 195] HLSTVVK </td><t开发&gt; 1 </td><td> 1 </td><td> 0 </td></tr><tr><td> GTELDQTHL [P S 198] TVVK </td><td> 10 </td><td> 7 </td><td> 5 </td></tr><tr><td> GTELDQTHLS [P T 199] VVK </td><td> 8 </td><td> 3 </td><td> 4 </td></tr><tr><td> G [P T 190] ELDQTHLS [P T 199] VVK </td><td> 0 </td><td> 1 </td><td> 2 </td></tr><tr><td> GTELDQTHL [P S 198] [P T 199] VVK </td><td> 0 </td><td> 8 </td><td> 4 </td></tr><tr><td> GTELDQTHLSTVVK </td><td> 46 </td><td> 26 </td><td> 48 </td></tr><tr><td> PTO肽187-202 </td><td colspan="3"></td></tr><tr><td> KGTELDQ [P T 195] HLS TVVK </td><td> 0 </td><td> 1 </td><td> 0 </td></tr><tr><td> KGTELDQTHL [P S 198] TVVK </td><td> 17 </td><td> 17 </td><td> 12 </td></tr><tr><td> KGTELDQTHLS [P T 199] VVK </t开发&gt; <td> 4 </td><td> 2 </td><td> 2 </td></tr><tr><td> KGTELDQ [P T 195] HLS [P T 199] VVK </td><td> 0 </td><td> 1 </td><td> 1 </td></tr><tr><td> KGTELDQTHL [P S 198] [P T 199] VVK </td><td> 0 </td><td> 7 </td><td> 5 </td></tr><tr><td> KGTELDQTHLSTVVK </td><td> 21 </td><td> 37 </td><td> 18 </td></tr><tr><td>肽187-202和188-202 </td><td> 83 </td><td> 88 </td><td> 50 </td></tr><tr><td>肽与2的磷酸化事件的百分比</td><td> 0％ </td><td> 26.9％ </td><td> 11.2％ </td></tr><tr><td> PTO序列覆盖</td><td> 78％ </td><td> 79％ </td><td> 82％ </td></tr></tbody></table> 表1 PTO的肽在激活信号 35S 的双磷酸化：。PRF-3xHA和35S：PTO-FLAG瞬时随EM表达PTY载体（EV），35S：AvrPto或35S：AvrPtoB在北本塞姆氏 。三天后浸润PTO-FLAG蛋白质的总量是用抗FLAG亲和基质免疫沉淀和解决在SDS-PAGE。的PTO上的胶体考马斯亮蓝染色凝胶可见光波段被切除并进行分析与质谱。确定与0的Pto肽，1，和2磷酸化事件的数目来表示。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>表缓冲区</td><td></td><td></td></tr><tr><td> L培养基</td><td>胰蛋白胨</td><td> 10克/升</td></tr><tr><td></td><td>酵母提取物</td><td> 5克/升</td></tr><tr><td></td><td>氯化钠</td><td> 5克/升</td></tr><tr><td></td><td> D-葡萄糖</td><td>为1g / L </td></tr><tr><td>农业渗透缓冲</td><td> 氯化镁 </td><td> 10毫米</td></tr><tr><td></td><td> MES &lt;/ TD&gt; <td> 10毫米</td></tr><tr><td></td><td>调节pH至5.5 </td><td></td></tr><tr><td></td><td>乙酰丁香酮（可选） </td><td> 150微米</td></tr><tr><td>缓冲液A </td><td>的Tris-HCl pH为7.5 </td><td> 150毫</td></tr><tr><td></td><td>氯化钠</td><td> 150毫</td></tr><tr><td></td><td> EDTA </td><td> 5毫米</td></tr><tr><td></td><td> EGTA </td><td> 2毫米</td></tr><tr><td></td><td>甘油</td><td> 5％（体积/体积） </td></tr><tr><td>缓冲液B </td><td>缓冲液A </td><td></td></tr><tr><td></td><td> PVPP </td><td> 2％（重量/体积） </td></tr><tr><td>缓冲液C </td><td>缓冲液B </td><td></td></tr><tr><td></td><td>二硫苏糖醇（DTT）的</td><td> 10毫米</td></tr><tr><td></td><td>植物蛋白酶抑制剂</td><td> 1％（体积/体积） </td></tr><tr><td></td><td>苯甲基磺酰氟（PMSF） </td><td> 0.5毫米</td&gt; </tr><tr><td></td><td> Calyculin一个</td><td> 50纳米</td></tr><tr><td></td><td>氟化钠（NaF） </td><td> 50毫米</td></tr><tr><td></td><td>钼酸钠（钠2的MoO 4） </td><td> 10毫米</td></tr><tr><td></td><td>原钒酸钠</td><td> 1毫米</td></tr><tr><td></td><td>冈田酸</td><td> 100纳米</td></tr><tr><td>缓冲液D </td><td>缓冲液C不PVPP </td><td></td></tr><tr><td> 5×SDS-PAGE上样缓冲液</td><td>的Tris-HCl pH 6.8的</td><td> 60毫米</td></tr><tr><td></td><td> SDS </td><td> 2％ </td></tr><tr><td></td><td>甘油</td><td> 0.15％ </td></tr><tr><td></td><td>溴酚蓝</td><td> 0.10％ </td></tr><tr><td></td><td> DTT（临用前加） </td><td> 50毫米</td></tr></tbody></table> 表2。缓冲液和介质配方。

Watch this Scientific Journal Video about Identification of Post-translational Modifications of Plant Protein Complexes at JoVE.com

Identification of Post-translational Modifications of Plant Protein Complexes

我们在这里描述的一个协议，用于植物蛋白质复合物的纯化和表征。我们证明了在一个复杂的免疫沉淀一个单一的蛋白质，这样我们就可以识别其翻译后修饰及其相互作用的合作伙伴。

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

identification-post-translational-modifications-plant-protein

Research

JoVE Journal

Biology

23.7K 次观看.  University of Warwick. 我们在这里描述的一个协议，用于植物蛋白质复合物的纯化和表征。我们证明了在一个复杂的免疫沉淀一个单一的蛋白质，这样我们就可以识别其翻译后修饰及其相互作用的合作伙伴。 

视频: Identification of Post-translational Modifications of Plant Protein Complexes

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

科研

生物学

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

检测组蛋白修饰在植物叶片

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

使用RIP-芯片的核糖核蛋白复合物的基因，小分子RNA和蛋白质组分的分离与鉴定细胞提取物的方法

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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 ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...