这项工作是由（在InvestigaciónCientíficaBASICA在Investigación连接国界德拉西恩西亚82614，153998，253549和178359以及118），并从提高妇女地位总署德从理事会全国国立科学城ŸTECNOLOGIA - 墨西哥（国家科学技术委员会和墨西哥）资助项目Asuntos去个人Académico-国立自治大学墨西哥（DGAPA，墨西哥国立自治大学; PAPIIT IN202616，IN203612）。

1.克隆和过度表达结构基因的脂肪酶预测<ol><li>使用聚合酶链反应（PCR）14和特异性寡核苷酸（ 表1）15，扩增感兴趣（smc01003，smc00930或smc00171）的基因，预测为脂肪酶编码或磷脂酶，从宿主生物的基因组DNA（ 即， 苜蓿根瘤菌 ）。 <ol><li>引入特定的限制性位点（有寡核苷酸的设计的序列）。消化与相应的限制性内切酶的扩增的DNA片段，并将其克隆到表达载体中，如PET系列16的质粒。 </li><li>验证为克隆基因的正确DNA序列后，转化载体至表达菌株如大肠杆菌 BL21（DE3）pLysS中16。 </li></ol></li><li>准备表达宿主大肠杆菌的过夜预培养大肠杆菌 BL21（DE3）PLYSS，窝藏与克隆基因或空载体的各个pET载体，在含有20ml的Luria BERTANI肉汤（LB）17 100毫升培养瓶中加所需的抗生素。培养细胞在30℃（或在从该脂肪酶源自细菌的通常生长温度）。 <ol><li>使用过夜预培养物，接种在2L培养瓶中500毫升预热的LB培养基（加所需抗生素）在620nm（OD 620）= 0.05，以获得一个初始光密度。跟随在的OD 620 = 0.3培养物和生长，添加异丙基β-D-硫代半乳糖苷（IPTG）至100μM的最终浓度，并在搅动下孵育在30℃，为期4小时。 </li><li>在温育期结束时，在5000 xg离心传送各培养到500ml的离心管并离心在4℃下30分钟。重悬细菌细胞沉淀在5ml悬浮缓冲液（ 例如 ，SMc00930-和SMc01003表达细胞在50mM的Tris-HCl pH 8.0的和SMc00171表达细胞在50mM二乙醇胺 - 盐酸pH值9.8）。在-80°C，直到使用储存细胞悬液。 </li></ol></li></ol> 2.准备无细胞蛋白提取物和确定蛋白质浓度<ol><li>除霜细菌细胞悬浮液并储存在冰上。传递细胞悬液通过冷压细胞三次，每2磅20000。离心5000 xg离心在4℃下除去完整细胞和细胞碎片30分钟。 </li><li>离心后，从上清液用于后续分析准备的100和500微升等分试样，并将其存储在-80℃直至使用。 </li><li>使用100微升等分试样中的一个由选择的方法或如所述18，以确定不同的无细胞提取物的蛋白质浓度。 </li></ol> 3.使用人工基质用于优化酶的（磷）的脂肪酶活动<ol><li>对于不同的酶活性的初始覆盖，使用通过水解产生有色产物人工基质，如对硝基苯酚（ 第 -NP）。 <ol><li>对于人工对硝基苯酯底物已经过优化的酶测定（概述的磷脂酶C PLCP（SMc00171），以及对预测的patatin启动状磷脂SMc00930和SMc01003），在表2中记载的使用移液的方案。 </li><li>当探索新的潜在的（磷）的脂肪酶，制备含有50mM Tris-盐酸，pH值8.5，100mM NaCl的，0.05％的Triton X-100，0.5mM的磷复合含有硝基苯基（ 对硝基苯磷酸盐的第一标准酶测定，二- 对硝基苯基酯，磷酸对硝基苯癸酸盐，或对硝基苯基棕榈酸酯），和无细胞蛋白提取物（检查1，3，10，30，100，300，和1000微克）以1:1的总体积毫升在1毫升塑料CUVEttes。 注意:下面的连续测定对硝基苯酯的水解时使用的碱性pH值（ 图1）。另外，使用单个时间点测定法的范围内的pH值，在温育期结束时加入NaOH以终止酶反应，并确保所有的p -NP存在于酚形式。 </li><li>按照时间过程为在30℃下在5分钟内于405nm的增加吸光度，因为p -NP的形成，在分光光度计。通过确定每时间吸光度的增加的初始斜率量化p -NP的最初线性形成。 </li><li>计算浓度（ΔC）的对p-NP使用朗伯-比尔定律（ΔA=εΔCd）1名的变化。 注:ΔA是吸光度确定的线性变化，ε是在各自的波长的摩尔消光系数（以M为单位-1 </s向上&gt; -1），d为光路（1厘米）的长度，和ΔC是浓度的（待确定的变化以M为单位）。 <ol><li>考虑到分析体积为1毫升，计算形成在p -NP量。 注:金额=浓度X卷。 </li><li>除以在它形成时形成在p -NP的量计算出的酶活性。通过蛋白质的这是负责生成该活动的量（以mg）除以酶活性确定特定的酶的活性。 </li></ol></li><li>通过比较蛋白质提取物挑起吸光度的变化，其中一个候选基因（smc00171，smc00930或smc01003）已表达了与港仅一个空向量提取物。 注意:为了继续执行下面的步骤，具体活动，造成其中的候选基因已被表达的蛋白提取物，应至少两次或间矿石比造成窝藏只有一个空的载体蛋白提取物的具体活动得到的数值。 </li><li>进一步的实验中，选择这些条件，其中含有硝基苯- 对化合物的水解是最小的与无细胞提取物（ 即，空载体）和的量，最明显形成p -NP的与p -nitrophenolate阴离子（ 图1）可以在蛋白质提取物中使用的，其中一个候选基因已被表达进行观察。 </li></ol></li><li>确定在3.1的初始酶活性后，通过改变pH值，缓冲液类型优化用于各酶的测定条件，缓冲强度的NaCl浓度，洗涤剂如Triton X-100和不存在的或不同的二价阳离子的存在。 <ol><li>对于不同浓度的各变量，确定特定酶的活性（见3.1.4.2）（获得最高数目限定的条件最大酶活性的）。使用的每个变量所遇到的最佳条件的组合来定义一个优化的酶测定，其中每个变量是存在于它的最佳浓度。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54613/54613fig1.jpg" /> 图1. 对 硝基苯酯在分光光度测定法（磷）的脂肪酶作为人工基质。当对硝基苯酯的水解，酸（R-OH）和对硝基苯酚（ 对 -NP）形成。由于pK a为= 7.2酚类的 H +从对 -NP解离，在pH&gt; 9.2的99％以上是在明亮的黄色p -nitrophenolate形式和18000 M -1 cm的摩尔消光系数- 1可以在波长使用<html>为游离对 -nitrophenolate 22的量化405纳米。当使用具有pH为8.5缓冲，吸收在400 nm和14500 M -1厘米的摩尔消光系数确定-1采用23。 <a href="http://ecsource.jove.com/files/ftp_upload/54613/54613fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 注:在已经确定的最佳条件为所关注的酶的活性，踏上寻求本脂肪酶的实/生理底物。原则上，利用2个，经常补充，方法来实现这个目标， 在体内方法或体外方法。 4.一脂肪酶的生理底物的体内鉴定ove_content"&gt;注意: 在体内的方式，表达了在宿主生物体8,11感兴趣的脂肪，才能随着时间的推移脂肪酶的表达是否改变了host&#39;s血脂登记在另一个体内的方法，生成突变体缺乏兴趣8,11的基因并研究其脂质分布是否是由野生型版本6,8,11是不同的。为了得到生物体的脂质分布的定量评估，一个简单的方法包括放射性标记细胞化合物，提取脂质，通过色谱法将它们分离，并量化放射性标记的分离脂质。 <ol><li>脂类的放射性标记。 <ol><li>在5毫升的所需培养基（复合培养基或定义基本培养基）的制备的兴趣（ 大肠杆菌或S.根瘤菌 ）有机体的过夜预培养，并在30℃下生长。 </li><li>从预培养，接种20毫升的股份有限公司在100毫升培养瓶中箱新鲜培养基，得到初始OD 620 = 0.3的培养。 </li><li>取培养物的等分试样（1ml）中在无菌条件下，并转移到14毫升无菌聚苯乙烯圆底管中。 </li><li>添加[1- 14 C]乙酸盐（每毫摩尔60毫居里）的1 ml培养1微居里。 </li><li>孵育在搅拌下在液体培养物在30℃，为期24小时。 </li><li>在温育期结束时，转移培养到1.5ml微量离心管中并离心以12,000 xg离心在室温下5分钟。 </li><li>重悬在100μl水中沉淀。在这一点上，存储所述细胞悬浮液在-20℃下或立即继续与极性脂质（第4.2节）的提取。 </li></ol></li><li>极性脂质的提取。 注:这里主要介绍的方法遵循布莱和代尔19日报道的过程。 <ol><li>以100微升水细胞停赛的ension，添加375微升甲醇:氯仿溶液（2:1;体积/体积）。 </li><li>涡旋30秒，并孵育在室温下5分钟。 </li><li>离心5分钟，在室温下以12000 xg离心。 </li><li>将上清转移至新的1.5 ml离心管中。 </li><li>加氯仿的125微升和125微升水，旋涡30秒。 </li><li>离心1分钟，在室温下12000 xg离心。 </li><li>下部氯仿相转移到新的管中，并干燥，用氮气流。 </li><li>溶解在100μl氯仿中干燥脂质:甲醇溶液（1:1;体积/体积）。 注意:在这一点上，5微升脂质溶液的等分试样可通过液体闪烁计数进行定量。 </li><li>对于薄层色谱（TLC）分析，干下来其余95微升用氮气流和在20μl氯仿中再溶解干燥的脂质:甲醇溶液（1:1;体积/体积）。使用3微升等分对于TLC分析。 </li></ol></li><li>通过薄层色谱（TLC）极性脂质的分离。 注意:根据所述脂质类进行分析，固体和流动相的不同组合可能被用于分离。此处带电极性脂质和另一个，更适合中性极性脂质，采用高性能薄层色谱法（HPTLC）硅胶铝板作为固相的典型分离，进行了概述。 <ol><li>由二维TLC（2D-TLC）带电极性脂质的分离。 <ol><li>在HPTLC硅胶铝板（10×10厘米），从板的边缘2厘米一角应用脂质样品的一个3微升等分试样。 </li><li>准备与在第一维混合用于分离的流动相（140毫升氯仿60毫升甲醇，和10毫升水）。 </li><li>大衣色谱纸TLC发展内部腔。 注意:这是为了确保该室的气体相将用于第一维流动相后迅速饱和（30分钟内）已经被添加到腔室和腔室已被封闭的玻璃板。 </li><li>制备并混合流动相（130毫升氯仿，50ml甲醇，和20ml冰醋酸），用于在所述第二维分离，并转移到一第二薄层显影室内部涂覆有色谱纸，让腔饱和。 </li><li>小心地将薄层硅胶铝板与干燥的脂质样品在封闭腔室中的第一维5转移到第一室和发展（ 即 ，执行色谱法）该板60分钟。 </li><li>从腔室中取出板，并让溶剂擦干在一个流罩30分钟。 </li><li> 90度相对于转动板到先前层析后，转移薄层硅胶铝板，在其上的脂质已经在一个维度被分离，给塞康ð腔和开发该板在第二维5 60分钟。 </li><li>除去从腔室的薄片，让该溶剂干燥掉在一个流罩至少2小时。 </li></ol></li><li>中性极性脂质的分离。 <ol><li>适用于启动从板的边缘2cm的薄层硅胶铝板脂质样品3微升等分试样。如果多个样品在一维色谱分析，保持的不同示例应用点之间至少1.5厘米的距离。 </li><li>制备并混合流动相（140毫升己烷，60毫升乙醚，和8ml乙酸），并转移到一个薄层显影室内部涂覆有色谱纸和覆盖有一个玻璃板，让腔饱和（30分钟）。 </li><li>与干燥的脂质样品转移薄层硅胶铝板到腔和发展板在封闭室30分钟。 </li><li>从室中取出板并让溶剂擦干在一个流罩2小时。 </li></ol></li></ol></li><li>定量分离极性脂类的可视化。 <ol><li>一旦开发了薄层片材干燥，以光激励发光（PSL）屏幕孵育它在一个封闭的盒3天。 </li><li>露出孵育屏幕到PSL扫描器并获取分离放射性标记的脂质的虚像。 </li><li>使用PSL软件20进行量化。 </li></ol></li><li>可视化和个人极性脂质类的隔离。 <ol><li>孵育发达型TLC片在以1g碘晶体的存在下层析室10分钟。 注:分居脂质化合物会溶解碘和出现褐色斑点。 </li><li>圈用铅笔斑点，把它们比作标准化合物（ 即 1,2- dipalmitoyl- SN -甘油，二棕榈-L-α-磷酸的相对移动（R F）phatidylcholine，DL-α-甘油一棕榈酸酯，或棕榈酸），并确定它们可能属于哪脂质类。 </li><li>在通风橱中，让碘在TLC板蒸发。 </li><li>用刮铲的帮助下，刮含有从纸张目的产物的硅胶，并用100微升的水和375微升甲醇中的混合物中提取的硅胶化合物:氯仿溶液（2:1;体积/体积）。 </li><li>根据布莱和代尔拔牙继续概述（4.2.2起）。 </li><li>商店纯化脂质类在100μl的氯仿:甲醇溶液:;在-20℃直到使用（1体积/体积）。 </li></ol></li></ol> 5.脂肪酶的生理底物的体外鉴别注意:在体外的方法，研究感兴趣的脂酶能否分离脂质或各个纯脂质的混合物转化成相应的hydrolysis产品在3.2定义为最佳的条件下进行。 <ol><li>使用酶分析吹打方案按表3为PC专用磷脂酶C SMc00171（见5.2），磷脂酶A（见5.3），以及DAG脂肪酶SMc01003（见5.4）的活动。 </li><li> PC专用磷脂酶C活性（ 表3）的测定。 <ol><li>到1.5 ml离心管中，加每分钟5,000计数单位共14个 C标记的PC（CPM）和Triton X-100的解决方案。 </li><li>混合和干燥在氮气流。 </li><li>添加二乙醇胺-盐酸，pH值9.8的缓冲，以及NaCl和的MnCl 2溶液和二次蒸馏水，以获得99.5微升的最终体积。涡旋5秒。 </li><li>加入0.5微升酶（5微克蛋白质）的（ 即，无细胞提取物，其中过表达SMc00171存在），以引发反应。简单地混合。 </li><li>孵育在30℃下4小时。 </li><li>停经反应另外的甲醇250微升和125微升氯仿。 </li><li>提取如先前所描述的脂质（见4.2）。 </li><li>单独的脂质通过一维（1D）-tlc（参见4.3.2和4.4），并通过PSL成像分析它们。 </li></ol></li><li>磷脂酶A活性的测定（ 表3）。 <ol><li>到1.5 ml离心管中，加总14 C标记的磷脂5000 CPM和Triton X-100的解决方案。 </li><li>混合和干燥在氮气流。 </li><li>为最终100μl的测定法中，添加的Tris-HCl，pH值8.5的缓冲，NaCl溶液和水。涡旋5秒。 </li><li>添加5微升的酶（50微克蛋白质）（ 即 ，无细胞提取物，其中过表达SMc00930或SMc01003存在）。 </li><li>孵育在30℃下5小时。 </li><li>停止通过加入甲醇250微升和125微升氯仿的反应。 </li><li>提取如先前所描述的脂质（见4.2），独立他们通过使用1D型TLC130毫升氯仿，50ml甲醇，和20ml冰醋酸作为流动相，并且通过PSL成像分析它们。 </li></ol></li><li>甘油二酯（DAG）脂肪酶活性的测定。 <ol><li> 14 C标记的DAG的制备。 <ol><li>放射性S.根瘤菌文化（见4.1），并提取极性脂质（见4.2）所描述的。独立S.利用在4.3.1中第二维分离所述的条件;（体积/体积20 130:50）， 根瘤菌总脂质提取物通过1D-TLC氯仿:甲醇乙酸。 </li><li>由碘染色可视化的PC和用铅笔标记磷脂酰胆碱（PC）的本地化。 </li><li>按照4.5中所述隔离放射性标记的PC。 </li><li>通过闪烁计数定量提取PC。 注:约32 CPM PC的预期。 </li><li>治疗用0.1ü 产气荚膜梭菌磷脂酶C的PC（250,000 CPM）在50毫米的Tris-HCl，pH值7.2，0.5％的Triton X-100和10mM氯化钙2为在100μl的总体积2小时，停止加入甲醇250微升和125微升氯仿的反应。 </li><li>因为他们通过1D-TLC（见4.3.2）以前和独立的描述中提取脂类。 </li><li>从硅石板隔离的二酰基甘油和通过闪烁计数量化（如在4.2中描述） </li></ol></li><li>二酰基甘油脂肪酶测定（ 表3）。 <ol><li>到1.5 ml离心管中，加入14 C标记DAG 5000 CPM和Triton X-100的解决方案。 </li><li>混合和干燥在氮气流。 </li><li>为最终100μl的测定法中，添加的Tris-HCl（pH为9.0）缓冲液，NaCl溶液和二次蒸馏水。涡旋5秒。 </li><li>发起通过添加5微升的酶（无细胞提取物的50微克蛋白质）的反应。 </li><li>孵育在30℃下4小时。 </li><li>停止通过加入250微升甲醇中的反应ð125微升如前所述氯仿和提取脂质（见4.2）。 </li><li>分析由1D-TLC（见4.1.3.2）和随后的PSL成像中性极性脂质。 </li></ol></li></ol></li></ol>

<ol>
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作者什么都没有透露。

在过去的20年中，许多生物基因组研究已经被测序，虽然丰富的基因组序列数据已经产生，功能解释相对滞后，因此阻碍了我们的基因组功能的认识。在基因组中的基因功能的基础上相似已知功能或保守的基序的发生基因经常被分配。然而，给定基因的精确功能往往不是公知的。尤其，预计酶的结构基因不能由组学技术容易地探索由于这样的事实，大部分酶催化涉及两个基板和两个产品的复杂的?...

基于甘油脂质，如三酰甘油和（甘油）磷脂构成了重要的可能是最知名的类脂质1。三酰基甘油（变量）的脂肪或油，其通常用作存储脂类，因此作为潜在的能量和碳的来源。标签可以通过脂肪酶，这是经常被生产菌分泌的消化外部变量并使它们可作为碳源退化。另外，脂肪酶已经被广泛地研究，多年来，由于其重要的生物技术应用2。 由于它们的两亲性质及其近圆筒状的形状，（甘油）磷脂展览成膜性，通常构成了双层膜3的主要类脂组分。在简单的微生物，如细菌大肠杆菌中，只有三个主要头组的变体，磷脂酰甘油（PG），心磷脂（CL），和phosphatidylethanolamine（PE）遇到，尽管人们应该知道，他们中的每一个都可以用在sn相当数量的不同脂肪酰基链的-1或SN -2位置引起大量不同分子种类4的被取代。其他细菌可能具有除了或代替其他磷脂。例如， 苜蓿中华根瘤菌 ，土壤细菌，其能够形成与豆科苜蓿（ 紫花苜蓿 ）一个固氮根瘤共生，包含除了PE的第二两性离子磷脂，磷脂酰胆碱（PC），5。另外，不含有脂质磷或甘油可能是细胞膜的两亲性并形成部分。例如，在磷限制生长条件下，在S根瘤菌 ，（甘油）磷脂主要由膜脂质不包含磷， 即，硫脂，鸟氨酸脂质和diacylglyceryl trimethylho取代moserine（DGTS）6。在细菌中，DGTS从二酰甘油（DAG）在一个两步通路7形成，但是对于DAG生成源不明确。脉冲追踪实验表明个人电脑可能是DGTS 8的前体，并使用在这个手稿，我们可以识别磷脂酶C（PLCP，SMc00171）中描述的方法，该方法是磷限制性的条件下形成并且可PC转换为DAG和磷酸8。 在另一项研究中，我们发现，酰基-CoA合成酶（FADD）S的缺陷型突变体进入生长9的固定相时的根瘤菌或大肠杆菌积累游离脂肪酸。尽管这些脂肪酸似乎是从膜脂质衍生自游离脂肪酸或解放出来的酶的确切来源没有已知的。再次，采用的策略在这个手稿概述，二patatin启动类10（磷）的脂肪酶（SMc00930和SMc01003），该对S中形成的游离脂肪酸贡献根瘤菌 11进行了预测。令人惊讶地，SMc01003使用的DAG作为底物将其转换为单酰甘油和最后甘油和游离脂肪酸11。因此，SMc01003是DAG脂肪酶（DGLA）。 虽然用于预测潜在（磷）存在一些算法脂酶12,13，它们的精确的功能和生理作用通常是不知道的。在这里，我们勾勒出一个协议，克隆和过度预测的或潜在的（磷）的脂肪酶。这份手稿解释酶检测如何开发和利用人工色底物的过度表达（磷）脂肪酶进行了优化。我们提供的例子，这些研究结果可能如何与优化的酶测定可遇到的实际（磷酸），酶底物，以及如何丰富我们的微生物生理学的理解。

<table><tbody><tr><td>Chloroform</td><td>JT Baker</td><td>9180-03</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Methanol</td><td>JT Baker</td><td>9070-03</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Acetic Acid</td><td>JT Baker</td><td>9507-05</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Hexanes</td><td>JT Baker</td><td>9309-02</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Diethylether</td><td>Sigma</td><td>32203</td><td>Enzymatic assays</td></tr><tr><td>bidistilled water</td><td>&amp;nbsp;ANY</td><td>&amp;nbsp;NA</td><td>Enzymatic assays</td></tr><tr><td>Tris Base</td><td>Sigma</td><td>T-1503</td><td>Enzymatic assays</td></tr><tr><td>HCl</td><td>Baker</td><td>9535-02</td><td>Enzymatic assays</td></tr><tr><td>NaCl</td><td>Baker</td><td>3624-01</td><td>Enzymatic assays</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X-100</td><td>Enzymatic assays</td></tr><tr><td>LB broth</td><td>ANY</td><td>NA</td><td>Bacterial growth, 10 g tryptone + 5 g yeast extract + 10 g NaCl per liter of bidistilled water</td></tr><tr><td>tryptone</td><td>Becton Dickinson and Company</td><td>211705</td><td>Bacterial growth</td></tr><tr><td>yeast extract</td><td>Becton Dickinson and Company</td><td>212750</td><td>Bacterial growth</td></tr><tr><td>TY broth</td><td>ANY</td><td>NA</td><td>Bacterial growth, 8 g tryptone + 3 g yeast extract + 66 mg CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O per liter of bidistilled water</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Baker</td><td>1332-01</td><td>Enzymatic assays</td></tr><tr><td>isopropyl-&amp;beta;-D-thiogalactoside (IPTG)</td><td>Invitrogen</td><td>15529-019</td><td>Bacterial growth</td></tr><tr><td>Diethanolamine</td><td>Sigma</td><td>D-8885</td><td>Enzymatic assays</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>221279</td><td>Enzymatic assays</td></tr><tr><td>Phospholipase A&lt;sub&gt;2&lt;/sub&gt; snake venom</td><td>Sigma</td><td>P0790</td><td>Enzymatic assays</td></tr><tr><td>Phospholipase C &lt;em&gt;Clostridium perfringens&lt;/em&gt;</td><td>Sigma</td><td>P7633</td><td>Enzymatic assays</td></tr><tr><td>Bis-&lt;em&gt;p&lt;/em&gt;-nitrophenyl phosphate</td><td>Sigma</td><td>07422AH</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl stearate</td><td>Sigma</td><td>N3627</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl dodecanoate</td><td>Sigma</td><td>61716</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl decanoate</td><td>Sigma</td><td>N0252</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl palmitate</td><td>Sigma</td><td>N2752</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl butyrate</td><td>Sigma</td><td>N9876</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl octanoate</td><td>Sigma</td><td>21742</td><td>Enzymatic assays</td></tr><tr><td>Acetic Acid, sodium salt [1-&lt;sup&gt;14&lt;/sup&gt;C]</td><td>Perkin Elmer</td><td>NEC084</td><td>Bacterial growth</td></tr><tr><td>dimethylsulfoxide (DMSO)</td><td>JT Baker</td><td>9224-01</td><td>Enzymatic assays</td></tr><tr><td>Aluminium HPTLC silica gel 60 plates. Silica gel HPTLC plates size 20 x 20 cm, 25 sheets.</td><td>Merck</td><td>105547</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Spectrometer UV/VIS Lambda 35</td><td>Perkin Elmer</td><td>NA</td><td>Enzymatic assays</td></tr><tr><td>Storm 820 Phosphorimager</td><td>Molecular Dynamics</td><td>NA</td><td>Photostimulable Luminescence scanner&amp;nbsp;</td></tr><tr><td>Multipurpose Scintillation Counter</td><td>Beckman Coulter</td><td>NA</td><td>Radioactivity Quantification</td></tr><tr><td>French Pressure Cell</td><td>ThermoSpectronic</td><td>NA&amp;nbsp;</td><td>Breakage of cells</td></tr><tr><td>chromatography paper 3MM Chr</td><td>Whatman</td><td>3030917</td><td>TLC analysis</td></tr><tr><td>&lt;em&gt;Sinorhizobium meliloti&lt;/em&gt; 1021our</td><td>reference 11</td><td></td><td>studied strain</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; BL21 (DE3) pLysS Competent cells</td><td>Novagen</td><td>69451</td><td>protein expression strain</td></tr><tr><td>pET9a vector</td><td>Novagen</td><td>69431</td><td>protein expression vector</td></tr><tr><td>pET17b vector</td><td>Novagen</td><td>69663</td><td>protein expression vector</td></tr><tr><td>sterile polystyrene round-bottom tube (14 ml) Falcon</td><td>Becton Dickinson</td><td>352057</td><td>radiolabeling of bacterial cultures</td></tr><tr><td>polypropylene microcentrifuge tubes (1.5 ml)</td><td>Eppendorf</td><td>30125.15</td><td>Enzymatic assays</td></tr><tr><td>1,2-dipalmitoyl-&lt;em&gt;sn&lt;/em&gt;-glycerol</td><td>Sigma</td><td>D9135</td><td>lipid standard</td></tr><tr><td>L-&amp;alpha;-phosphatidylcholine, dipalmitoyl</td><td>Sigma</td><td>P6267</td><td>lipid standard</td></tr><tr><td>DL-&amp;alpha;-monopalmitin</td><td>Sigma</td><td>M1640</td><td>lipid standard</td></tr><tr><td>palmitic acid</td><td>Sigma</td><td>P0500</td><td>lipid standard</td></tr></tbody></table>

defining substrate specificities for lipase and phospholipase candidates

微生物产生的那些，以使可用于生物体外部基板分泌（磷）的脂酶广泛光谱。另外，其他（磷酸）脂肪酶可以在物理上与生产菌引起的内在脂质的营业额，并经常引起细胞膜的重塑有关。虽然势（磷）的脂肪酶可以用数目的算法时该基因/蛋白质序列可用来预测，经常没有获得的酶的活性，底物特异性，和潜在的生理功能的实验证据。这个手稿描述的测定条件与未知底物特异性，以及如何利用这些优化条件下在搜索用于相应（磷）的脂肪酶的天然底物准（磷）的脂肪酶的最优化。采用人工发色底物，如对硝基苯衍生物，可有助于检测小为在标准条件下的预测（磷）的脂肪酶的酶活性。具有遇到这样的次要的酶活性，酶测定法的不同参数可以在为了获得人造基底的更有效的水解而变化。已确定在其下的酶工作良好的条件之后，各种潜在天然底物应测定其降解，其可以采用不同的层析方法进行随后的处理。底物特异性为新酶的定义，通常提供假设这些酶，然后可通过实验测试的潜在生理作用。遵循这些指导原则，我们能够确定降解磷脂酰胆碱磷酸胆碱到甘油二酯和，在膜中生长时的磷限制条件细菌根瘤菌重塑的关键一步磷脂酶C（SMc00171）。两个预测patatin-就像有机体一样的磷脂酶（SMc00930和SMc01003），我们可以重新确定自己的底物特异性和澄清SMc01003是甘油二酯脂肪酶。

PC专用磷脂酶C SMc00171的活性的双- 对 硝基苯磷酸酯 从大肠杆菌获得的无细胞提取物大肠杆菌 BL21（DE3）×pLysS中，其中有smc00171表达，进行了研究它们对水解二- 对硝基苯基磷酸酯的能力，使用分光光度酶促测定法中，测量形成p -NP。没有水解活性是?...

Watch this Scientific Journal Video about Defining Substrate Specificities for Lipase and Phospholipase Candidates at JoVE.com

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

定义底物特异性的脂肪酶和磷脂酶考生

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

defining-substrate-specificities-for-lipase-phospholipase

Research

JoVE Journal

Biochemistry

14.9K 次观看.  Universidad Nacional Autónoma de México. Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

视频: Defining Substrate Specificities for Lipase and Phospholipase Candidates

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

磷脂Scramblase活动的基于荧光的测定法

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

科研

生物化学

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Channels for the transportation of water molecules in enzymes influence active site solvation and catalysis. Herein we present a protocol for the engineering of these additional catalytic motifs based on in silico computer modeling and experiments. This will enhance our understanding of the influence of solvent dynamics on enzyme catalysis.

揭开熵率的加速溶剂动力学的膜酶诱导

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

化学

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

使用微尺度热泳测量蛋白质-脂质相互作用

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

生物工程

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, nutrient fortification, food allergies, and digestive diseases. The ability of an emulsion to provide the desired functionality, namely, reaching a specific site within the gastrointestinal tract, inhibiting/retarding lipolysis, or facilitating digestibility, ultimately depends on its susceptibility to enzymatic degradation in the gastrointestinal tract. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers, where the emulsifiers stabilize the emulsion and protect the encapsulated compound. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the evolution of those interfacial layers as they are subjected to different phases of gastrointestinal digestion. A pendant drop surface film balance implemented with a multi-subphase exchange allows for simulating the in vitro digestion of emulsions in a single aqueous droplet immersed in oil by applying a customized static digestion model. The transit through the gastrointestinal tract is mimicked by the subphase exchange of the original droplet bulk solution with artificial media, mimicking the physiological conditions of each compartment/step of the gastrointestinal tract. The dynamic evolution of the interfacial tension is recorded in situ throughout the whole simulated gastrointestinal digestion. The mechanical properties of digested interfaces, such as interfacial dilatational elasticity and viscosity, are measured after each digestion phase (oral, gastric, small intestine). The composition of each digestive media can be tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies and infant digestive media. The specific interfacial mechanisms affecting proteolysis and lipolysis are identified, providing tools to modulate digestion by the interfacial engineering of emulsions. The obtained results can be manipulated for designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

A pendant drop surface film balance implemented with a multi-subphase exchange, nicknamed the OCTOPUS, allows for mimicking digestive conditions by the sequential subphase exchange of the original bulk solution with simulated gastrointestinal fluids. The simulated in vitro digestion is monitored by recording in situ the interfacial tension of the digested interfacial layer.

体外 通过模拟胃肠液的多亚相交换 在 单个液滴中消化乳液

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

定义底物特异性的脂肪酶和磷脂酶考生

摘要

探索更多视频

此视频中的章节

磷脂Scramblase活动的基于荧光的测定法

揭开熵率的加速溶剂动力学的膜酶诱导

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

使用微尺度热泳测量蛋白质-脂质相互作用

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

体外通过模拟胃肠液的多亚相交换在单个液滴中消化乳液

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

定义底物特异性的脂肪酶和磷脂酶考生

摘要

探索更多视频

此视频中的章节

磷脂Scramblase活动的基于荧光的测定法

揭开熵率的加速溶剂动力学的膜酶诱导

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

使用微尺度热泳测量蛋白质-脂质相互作用

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

PIP-on-a-chip:蛋白质 - 磷酸肌醇相互作用的无标记研究

体外 通过模拟胃肠液的多亚相交换 在 单个液滴中消化乳液

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

体外通过模拟胃肠液的多亚相交换在单个液滴中消化乳液