We thank Dr. Ben Nacev for advice on design of the photoaffinity labeling protocol, Dr. Wei Shi for synthesizing the itraconazole photoaffinity probe, Dr. Yongjun Dang for advice on affinity pull-down experiments, and other members of the J.O.L. laboratory for helpful comments and support. This work was supported in part by a PhRMA Foundation Fellowship in Pharmacology/Toxicology (to S.A.H.); National Cancer Institute Grant R01CA184103; the Flight Attendant Medical Research Institute; Prostate Cancer Foundation (J.O.L.); and the Johns Hopkins Institute for Clinical and Translational Research, which is funded in part by Grant UL1 TR 001079 from the National Center for Advancing Translational Sciences (NCATS).

注:此协议是改编自麦金农和汤顿10在活细胞中的使用。 1.培养细胞的制备<ol><li>制备无菌6厘米细胞培养皿为所需的样本数（见下文）。 注:细胞的一个培养皿每个处理条件下使用，但如果需要更多的蛋白2或3菜肴每种条件进行制备和UV照射后组合。 <ol><li>制备的细胞中的至少3菜肴;阴性对照与DMSO只（D）中，只有探头治疗（P）和探头+竞争对手（C）。 </li><li>任选地，准备4 个菜作为无紫外线控制，与探针进行处理，但不进行UV光。 注意:如果多个竞争者分子进行测试，如感兴趣的小分子的不同的类似物，制备额外竞争者板是必要的。 </li></ol></li><li> HEK293T细胞添加到培养皿在4毫升培养基中。使用350万Ç每盘厄尔。 注:应确定细胞的数目对于每个细胞类型，使得细胞在实验开始时几乎100％汇合，以达到最大的蛋白质产量。对于HUVEC，用每盘30万细胞。一个好的近似是一个融合的15厘米菜细胞的1/10。 HEK293T细胞应该在被培养的Dulbecco氏改良的Eagles培养基补充有10％胎牛血清（FBS）和1％青霉素/链霉素。 </li><li>返回细胞培养培养箱中（37℃，5％CO 2）过夜。 </li></ol> 2.细胞和光交联处理<ol><li>第二天，预等分下列药物入1.5ml微量管: <ol><li>对于第一次治疗，加入4微升DMSO中至2管和4微升10毫竞争者（即未改性的母体化合物）的1管。 </li><li>用于第二处理中，添加16微升的DMSO以1管和16微升的50＃181，M光亲探至2管。 注:最佳浓度可能是不同的探针（见讨论）不同，但在200-500纳米之间，是一个很好的起点（这里我们使用200纳米）。竞争者的浓度应超过使探针至少20倍（这里我们使用10μM）的。 DMSO的终浓度应在所有平板相等，并应不超过0.5％（20微升体积）。 </li></ol></li><li>使细胞板到细胞培养罩并且从步骤2.1.1添加预先等分的药物如下:吸1毫升培养基，再悬浮预等分药物在媒体上，并轻轻地加入它回到板降明智的。二甲基亚砜加入到二甲基亚砜（D）的板和探针（P）的板，和竞争添加到竞争（C）的板。返回所述板的培养孵化30分钟。 </li><li>在调暗培养罩灯，从步骤2.1.2添加-等分预探针来探测（P）和竞争（C）的板，并添加DMSO中的二甲基亚砜（D）的板，如上述。返回所述板的培养孵化1小时。 </li><li>在培育期间，请准备以下物品;冰托盘可以容纳所有的板块;冰冷的裂解缓冲液（PBS [pH为8.5] + 1×蛋白酶抑制剂鸡尾酒[完整无EDTA，根据水中的50倍原液稀释]; 200微升/板）;离心管标记为D，P或C的每个的不同的处理条件下，在冰上。 </li><li> 1小时培养结束前15分钟，成立了紫外灯（365纳米），在寒冷的房间，并打开它热身灯泡。 </li><li>与探针孵育1小时后，放置碗碟冰上。清洗细胞轻轻用5ml冰冷的PBS（pH 7.4）中以除去过量的探针。再覆盖用4ml冰冷的PBS中的细胞。 </li><li>将集中在冰袋上方的紫外灯在3厘米至最小化从灯加热单元的菜。照射3分钟。取下菜冰托盘并重复所有样品。 </li><li>一个压脚提升照射，从细胞吸出PBS并添加冰冷的PBS（pH为8.5）与蛋白酶抑制剂对各板的200微升。从使用橡胶刮刀，并转移到冰上预标记的微量离心管板分离细胞。 </li><li>加入SDS至0.4％的终浓度（10微升10％SDS的入250微升样品的）。 注意:SDS粉是危险的。避免穿了鼻子和嘴口罩吸入SDS粉末。 </li><li>裂解的细胞通过超声处理该悬浮液为10个脉冲（输出1，占空比30％）和第二轮10的脉冲之前在冰上孵育1分钟。 </li><li>如果需要的话，除去2微升悬浮液并在光学显微镜下检查，以确保完全细胞裂解。 </li><li>沸腾的热板上设置为95℃5分钟，以完成细胞裂解和变性的所有蛋白质的样品。 </li><li>使用分光光度洗涤剂相容蛋白测定中，如第测定各样品中的蛋白质浓度ÈDC蛋白测定19，根据制造商的说明，用设定在750纳米测量吸光度分光光度计。 </li><li>通过根据需要加入的PBS pH值8.5±0.4％SDS的正常化蛋白质浓度2.5毫克/毫升（或如果浓度低于2.5毫克/毫升，正常化到具有最低蛋白质浓度样品）（例如，如果样品是5在250微升毫克/毫升，加入250微升的PBS pH 8.5的0.4％的SDS，得到2.5毫克/毫升终浓度）。 </li></ol> 3.附件荧光标签通过点击化学的标记蛋白的可视化<ol><li>除去将40μl细胞裂解物并转移到一个新的离心管中。保持其余裂解物与生物素 - 叠氮化物（步骤4）的反应。 </li><li>添加下列试剂依次是:0.2微升氟叠氮（1毫米原液在DMSO），0.58微升TCEP（100毫米股票在水中4当量的NaOH加入，新鲜制备）和3.38微升TBTA（1。在4 7毫米股票:叔丁醇到DMSO 1的比例）。旋涡混合。 </li><li>添加1.14微升的CuSO 4 -5H 2 O（50毫在水中）以开始反应。短暂涡旋并孵育在室温下在黑暗中30分钟。 </li><li>加入50μl2×SDS样品缓冲液以终止反应。此时，直接在SDS-PAGE凝胶上20运行样品最好的结果，或在-20℃过夜如果需要存储。 </li><li>如果冷冻样品装载到凝胶之前，在95℃再加热5分钟。 注:由于目标蛋白质的分子量通常在此阶段不公知的，最好是运行2凝胶具有不同的丙烯酰胺浓度（ 即 ，8％和15％），或宽分子量范围的梯度凝胶（即 4-15％），以确保完全分子量覆盖。 </li><li>当染料前沿到达凝胶的末端，继续运行凝胶额外5分钟，以确保所有的过量unreacte的ð氟叠氮化合物已经完全退出了凝胶。 </li><li>与DDH 2 O的取出凝胶的容器和孵育10分钟，轻轻摇动，洗去所有多余的氟，叠氮化物。 </li><li>将凝胶在玻璃板上，并使用根据制造商的说明一台风荧光凝胶扫描仪扫描凝胶。 </li></ol> 4.附生物素标记的通过点击化学的标记蛋白的亲和纯化<ol><li>从步骤3.1剩余裂解物，使用的最大量的蛋白质归一化之后，使得所有的样品是相同的体积（例如，如果标准化后到2.5毫克/毫升所得的样品体积是500，550，和600微升，使用500每微升）。 </li><li>通过加入50微升的高容量链霉琼脂糖珠，预洗涤2次，用PBS（pH 7.4）中预清除裂解物。孵育1小时在4℃轮换。 </li><li>沉淀通过离心珠在1000 xg离心3分钟。除去叔他上清液在冰上一个新的离心管并弃去珠。 </li><li>除去DMSO样品1-2％到标有"输入"的新管中。添加2×SDS样品缓冲液并储存的等效体积在-20℃。 </li><li>每500微升裂解液，添加下列试剂:1.38微升生物素 - 叠氮化物（10毫米股票DMSO），5.5微升TCEP，和32.5微升TBTA。旋涡混合。 </li><li>加入11微升硫酸铜每500微升裂解液和旋涡简要-5H 2 O。在室温下孵育30分钟。 </li><li>加冷却到-20℃的丙酮4样品体积。涡旋样品，并在-80℃孵育过夜以完全沉淀的蛋白质和除去未反应的生物素 - 叠氮化物。 </li><li>离心机在17000×g离心15分钟，在4℃下将样品以沉淀沉淀的蛋白质。 </li><li>完全吸出上清液，并在150μl的PBS（pH为7.4）+ 1％SDS通过超声处理resolubilize的蛋白质。 </li><li&gt;添加600μl的PBS（pH 7.4）中，以SDS浓度稀释至0.2％。 </li><li>样品添加到30微升洗涤的预高容量链霉琼脂糖珠，并在4℃下孵育1小时以转动。 </li><li>沉淀通过离心珠在1000 xg离心3分钟。吸出含有未结合的蛋白，弃上清。 </li><li> 1毫升洗涤缓冲液（400毫摩尔NaCl，50毫摩尔Tris，0.2％SDS，pH7.4）中添加到珠子和孵化在室温下用旋转5分钟。 </li><li>重复步骤4.12和4.13的3倍。 </li><li>从珠粒完全吸出洗涤缓冲液，并加入30微升2×SDS样品缓冲液中。 </li><li>孵育5分钟95℃的加热块上，以从珠释放的蛋白质。 </li><li>离心1分钟的珠粒在室温下以13000 xg离心。 注意:在这一点上，可将样品储存在-20℃，直至准备好运行的SDS-PAGE。如果冷冻样品，在使用前在95℃下再加热5分钟。 </li><li>小心吸取了含有的蛋白质珠样品缓冲液，并加载到SDS-PAGE 20（见下文重要的考虑因素）。珠可以保存如有必要，以后再沸腾。 注:对于由银染色蛋白质检测，使用1mm厚的凝胶，得到更好的结果。如果频带要被切出质谱（MS）分析的银染凝胶，制备从无菌溶液新鲜的所有试剂，并在凝胶上采样之间留下一个空孔。为从探头车道切割每个频带，一个平行切片应从二甲基亚砜车道可采取使背景蛋白可以减去。对于通过蛋白质印迹蛋白的ID验证，这是至关重要的运行SDS-PAGE凝胶时也从步骤4.4加载的输入采样。 </li><li>遵循两种银染21或印迹22检测靶蛋白（S）的标准协议。 </li></ol>

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The authors have nothing to disclose.

不同的方法来鉴定小分子的目标，大致可分为两类:自上而下，其中药物的细胞表型被用于基于其已知功能来缩小其潜在的目标，或自下而上，其中目标通过化学或遗传方法3直接识别。自上而下或表型的研究可以识别受药物的某些细胞过程（ 例如 ，转录/翻译/ DNA合成，细胞周期阻滞，信号通路的激活/禁止等 ）可能背后的小分子的最终表型，这帮助的潜在目标的列表缩小到?...

生物活性的小分子基本上通过与相互作用并改变一个或多个"目标"的分子，最常见的蛋白质的功能，在小区工作。在药物发现中，当活性化合物是通过表型筛选发现，该化合物的分子靶（多个）的识别是关键的，不只是对于理解动作和化合物的潜在副作用的机制，但也潜在发现新的生物学基础的疾病模型，从而为新机制类治疗1发展道路。虽然不要求目标识别的一种药物用于治疗，近年来出现了越来越认识到，新的候选药物更容易在临床试验中取得成功，并因此产生更好的投资回报，如果一个有效的目标是已知2。因此，一直存在的方法用于鉴定小的兴趣日益增​​长分子靶蛋白。 一个经典的目标识别试验通常依赖于亲和纯化，其中感兴趣的小分子被固定在树脂并用全细胞裂解物，在这之后未结合的蛋白质被冲走，而其余的蛋白质被洗脱，并确定3温育。虽然这一技术已被用来鉴别许多小分子4的目标，它不适合作为用于几个原因的通用目标ID方法。首先，目标蛋白质必须保留在细胞裂解其天然构象，以保留其结合到小分子的能力。这可以是膜蛋白，其通常经历构象变化从它们的天然环境中取出后，或简单地聚合和溶液中沉淀出来尤其成问题。第二，小分子必须用化学方法以这样的方式，它可以固定到树脂改性，同时保持其与靶蛋白结合的能力。一旦被固定于树脂深结合口袋可能因此变得不可访问的小分子。第三，结合亲和力必须足够高，使得在洗涤步骤的相互作用被维持，使得低亲和力相互作用有挑战性的鉴定。如pH值，离子浓度，或其他内源性分子的存在第四，环境条件可在细胞内空间变化，并且有时会发生药物 - 靶相互作用的先决条件。因此，找到正确的条件，以允许和维持结合细胞外可能需要反复试验的一个显著量。 光亲和标记通过允许共价小分子和细胞的天然环境中的目标的结合规避这些问题。而不是固定的小分子要大笨重的树脂，在分子，而不是化学修饰安装两个小功能摹roups:一个光活化部分，其允许共价交联到靶蛋白时的光的特定波长照射，和一个报告基团，它允许待检测的靶蛋白，并随后分离。活细胞用的光亲和探针处理，探针结合和共价交联到靶蛋白，和探针 - 蛋白质复合物，然后分离完好。探针结合到靶的特异性由并联，其中过​​量的母体化合物的用于竞争远到靶蛋白的探针的结合进行竞争实验证明。 光亲和探针的设计和合成变化很大从一个小分子到另一个，并且不会在这个协议中支付;然而，关于这个问题的几个优秀的讨论已经发表5-9。主要考虑的是，该探针保留母体化合物的生物活性，因此presumabLY结合相同靶（多个）。结构 - 活性关系（SAR）研究必须进行，以确定其中分子的部分可以在不活性的损失进行修改。多种不同化学基团的已被用作可光活化交联剂，包括diazirine，二苯甲酮和芳基叠氮化物，它们各自具有优点和缺点10。同样地，也有已经用于分离探针结合蛋白多记者标记。报告基团可以是对自己的，功能如常用的生物素或荧光标记，或可能是需要的光交联步骤，其中有被小，因此不太可能损害生物活性11的优点随后进一步官能的前体。 在这个协议中，我们已经使用含有diazirine光致团的光亲探针，以及用于报告基团的通过的Cu（Ⅰ）的附接末端炔-catalyzED叠氮炔夏普勒斯-胡伊斯根环（或点击）反应12-15。特区研究，探针的设计和合成，以及这些研究的结果已被别处16-18出版。

<table border="0" cellpadding="0" cellspacing="0" width="1169">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tris(2-carboxyethyl)phosphine (TCEP)</td>
			<td style="width:188px;">Life Technologies</td>
			<td style="width:117px;">20490</td>
			<td style="width:481px;">Make fresh day of use. Prepare 100 mM stock in water with 4 eq NaOH.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA)&#160;</td>
			<td>AnaSpec</td>
			<td>63360-50&#160;</td>
			<td>Prepare 1.7 mM stock in a 4:1 ratio of t-butanol to DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Copper Sulfate (CuSO4-5H2O)</td>
			<td>LabChem, Inc.</td>
			<td>LC13440-1&#160;</td>
			<td>Prepare 50 mM stock in water, store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin-azide</td>
			<td>Click Chemistry Tools</td>
			<td>AZ104-100</td>
			<td>Prepare 10 mM stock in DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647-azide&#160;</td>
			<td>Life Technologies</td>
			<td>A10277</td>
			<td>Prepare 1 mM stock in DMSO, store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">365 nm UV lamp</td>
			<td>Spectroline</td>
			<td>FC100</td>
			<td>UV-blocking glasses should be worn while operating.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease inhibitor tablets, EDTA-free</td>
			<td>Roche Life Science</td>
			<td>11873580001</td>
			<td>Prepare 50x solution in water and store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>Branson</td>
			<td>Sonifier 250</td>
			<td>Set to output 1, duty 30%.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent gel scanner</td>
			<td>GE Healthcare Life Sciences</td>
			<td>FLA 9500</td>
			<td>Use red laser to detect Alexa-fluor 647.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Detergent-compatible Dc protein assay kit</td>
			<td>Bio-Rad</td>
			<td>5000112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Capacity Streptavidin Agarose beads&#160;</td>
			<td>Life Technologies</td>
			<td>20359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagles Medium, low glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>11885092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, qualified</td>
			<td>ThermoFisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptamycin solution</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS Sample Buffer (2X)</td>
			<td>ThermoFisher Scientific</td>
			<td>LC2676</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

这里示出的结果与抗真菌药伊曲康唑，它的使用，已经出版16的光亲和性探针获得。这些结果表明使用活细胞光亲和标记技术在成功地识别的主要依他康唑结合蛋白作为35kDa的膜蛋白电压依赖性阴离子通道1（VDAC1）。 上述方案是使用用于photolabeling和未修饰的伊曲康唑作为竞争分子的依他康唑的探针在HEK293T细胞中进行。...

Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

identification-small-molecule-binding-proteins-native-cellular

Research

JoVE Journal

Biochemistry

12.5K 次观看.  Johns Hopkins University School of Medicine. We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis. 

视频: Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

科研

生物化学

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Here we present a protocol to extract, resolve and identify mitochondrial supercomplexes which minimizes exposure to detergents and Coomassie Blue. This protocol offers an optimal balance between resolution, and preservation of enzyme activities, while minimizing the risk of losing labile protein-protein interactions.

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

摘要

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BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

肾小球的分离及肾小球细胞表面蛋白的活体标记

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

成熟水稻的横向切片（米苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

摘要

探索更多视频

此视频中的章节

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

肾小球的分离及肾小球细胞表面蛋白的活体标记

利用单击化学辅助 rna-路特捕获 (无花果) 策略捕获和鉴定 rna 结合蛋白

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

成熟水稻的横向切片（米苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核