The authors thank Dr. Dario Bonetta for providing Arabidopsis thaliana seeds and for technical assistance in regards to the triple response assay, as well as Simone Quaranta for help with FT-IR. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC-DG) to JLS, an Ontario Graduate Scholarship (OGS) to RVA, and a Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST) to AJV.

1.化学品<ol><li>制备500mM的安排（144.49克/摩尔）的溶液中，并同时包含500 mM氯化钠（58.44克/摩尔）中的溶液和500毫的NaH 2 PO 4·H 2 O（137.99克/摩尔）中酸化（pH 2.5）超纯水或0.1N HCl中。混合使用一个旋涡，直到解决方案是明确的。 </li><li>连续稀释（10倍）的500mM的溶液在相同的溶剂，得到的5mM和50mM的股票。 </li><li>制备的1-氨基环丙烷甲酸（ACC;101.1克/摩尔）的10mM溶液在超纯水。 </li><li>过滤消毒在-20°C股票的解决方案和存储等分。 </li><li>过滤消毒纤维素酶。商店等分在4℃。 </li></ol>从2-氯乙基酸分解2.验证乙烯生产:三重反应分析<ol><li>表面消毒拟南芥使用气相法生态型哥伦比亚种子: 注意:以下步骤产生Ť好氧氯气。进行种子消毒在通风橱中。 <ol><li>获得的可密封容器足够深以容纳种子灭菌的250ml玻璃烧杯中，并将其放置在通风橱中。 </li><li> A.添加拟南芥种子离心管，并将管在机架中。将装有开管到密封的容器架。 注意:不要填写个人管在半满，使氯气渗透到较低的种子。 </li><li>放置装有在可密封容器100毫升市售漂白剂的250毫升烧杯中。小心加入3毫升浓盐酸于漂白，并立即封住其盖子的容器。 注意:漂白和HCl反应而产生，其作用进行表面消毒的种子有毒的氯气。 </li><li>孵育在氯气存在下的种子在通风柜4小时。 注:较长的消毒时间会降低发芽效率。 </li><li>灭菌后，以便Chlorine气体在通风柜泄至少1小时，然后密封该离心管中。丢弃之前离开的漂白剂和盐酸混合物，在通风柜至少24小时。 注意:灭菌的种子可在室温下保存立即使用。 </li><li>保持种子在4℃下长期贮存。打开微量离心管，以防止冷凝之前带来种子至室温。商店的种子在黑暗中。 </li></ol></li><li>在4个部门的培养皿（90×15毫米）准备琼脂平板: <ol><li>准备A.110毫升生长培养基拟南芥苗:1×Murashige和含有1％斯库格（MS）的基础培养基15（4.33克/升）（重量/体积）蔗糖和0.8％（重量/体积）的琼脂。调整用NaOH的MS培养基至pH 6。 </li><li>制备100毫升CEPA分解细菌生长培养基:施拉姆和Hestrin含16 1.5％（SH）培养基（重量/体积）的琼脂。调整用NaOH的SH培养基至pH 7。 </li><li>通过高压灭菌和消毒坦佩媒体R IN 55℃水浴。 </li><li>一旦MS琼脂回火，加40毫升的无菌瓶中。为了准备A.阳性对照生长培养基拟南芥幼苗，补充40毫升一份的MS琼脂的40微升10毫ACC，得到10μM的ACC的终浓度。 </li><li> 5ml培养基添加到的扇形培养皿中（ 图1）相应的象限，并允许在琼脂固化。准备好所有板块一式三份。 注意:CEPA不被添加到生长培养基中，直到在后面的协议。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54682/54682fig1.jpg" /> 图 1: 用于与CEPA的三重反应实验琼脂平板上的安装示意图说明具体为阴性对照组（A），阳性对照（B）的象限，和实验板（C）。这个数字已经从Augimeri和带9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig1large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <ol start="3"><li>分层A.拟南芥种子保证同步萌发: <ol><li>加入大约50 A.拟南芥种子到包含MS或MS + ACC琼脂每个象限。确保种子均匀分布以促进苗去除和分析。 </li><li>孵育含板种子在黑暗中在4 °下3-4天。 </li></ol></li><li>分解CEPA对细菌生长培养基（SH）和执行三重反应分析: <ol><li>分层后，暴露的种子，以荧光灯2小时。 </li><li>蔓延10微升的500mM CEPA原液在含SH琼脂试验板的象限（pH值为7; 图1），以获得最终CEPA浓度1毫米。 </li><li>与密封实验薄膜板材和铝箔盖创造的种子黑暗的环境。 </li><li>通过在23℃下与琼脂面朝下在黑暗中温育板3天发芽种子。 注意:板可堆叠，但阴性对照应放置在底部，因为乙烯是比空气轻。 </li></ol></li><li>分析三重反应分析数据: <ol><li>用火焰灭菌的镊子，从对应于解剖或数字的USB显微镜下的每个处理和视图板除去单苗。确保苗的底部对齐，并且照片。 </li><li>取下每个象限30苗（每生物重复60苗每处理180苗）。对齐表面上的黑色背景和照片用尺子。 </li><li>测量使用ImageJ软件17重复苗的下胚轴长度（mm）。通过点击并拖动*直设置比例*工具选择为10毫米的长度。选择"设置刻度"的"分析"选项卡下的和已知的距离设置为10使用*分段*工具，单击并拖动来选择胚轴和按M键的长度测量距离。比较使用单向ANOVA和Tukey多重比较检验生物学重复的平均值。差异被认为是显著如果p &lt;0.05。 </li></ol></li></ol>整个细菌生长pH值为3分析<ol><li>一式三份成长和量化Komagataeibacter xylinus发酵剂: <ol><li>接种K的单菌落xylinus到5ml的SH培养基（pH为5），补充了0.2％（体积/体积）过滤灭菌纤维素酶。孵育在30℃下以150rpm搅拌培养物，直到OD达到0.3〜0.4 600（约72小时）。 </li><li>通过离心收获发酵剂（2,000 xg离心; 4℃; 10分钟）。用5毫升的无菌0.85％（重量/体积）NACL溶液，洗涤细胞两次，重悬细胞沉淀。保持细胞在冰上。 </li><li>量化使用佩特罗夫，豪塞尔计数室细胞。 </li></ol></li><li>接种培养pH值分析: <ol><li>增加150个10ml补充有0.2％（V / V）过滤灭菌纤维素到十二500毫升烧瓶用铝箔盖的SH培养基（pH 7）的。与接种瓶K. xylinus起始培养以10 5个细胞/ ml的浓度。用三个发酵剂，每次准备治疗三次生物学重复。 </li><li>补充培养物用300μl的5，50或500mM的安排储备溶液，以获得最终CEPA浓度的0.01，0.1和1.0毫米。补充用300μl用于溶解CEPA溶剂的未处理的对照培养物。 </li><li>通过紧密胶带箔盖的烧瓶密封烧瓶中并在150rpm下搅拌，在30℃孵育培养物14天。 </li></ol></li><li>每一天，asepticaLLY通过离心除去从每个烧瓶中并沉淀格5 1ml样品（2,000 xg离心; 4℃; 10分钟）。转移上清液至一个干净的试管，并测量使用pH计各生物复制的pH。 </li><li>通过作图的生物学重复的均值pH值分析时程数据。 注意:重要的是，培养物的pH不低于3.5; 5个培养物pH显著降低乙烯释放从安排，和pH值低于3.5完全抑制乙烯释放。 </li><li>为了控制氯和磷酸盐水平，执行使用0.01,0.1相同的实验，并用5，50和500毫米的股票，分别的NaCl的NaH 2 PO 4溶液的1.0mM的。 </li></ol> 4.菌落形态<ol><li>成长K. xylinus发酵剂培养物一式三份: <ol><li>接种K的单菌落xylinus到5ml的SH培养基（pH为5），补充了0.2％（体积/体积）过滤灭菌纤维素酶。 Incuba德培养在30℃下以150rpm搅拌直到0.3至0.4的OD 600达到（约72小时）。 </li><li>通过离心收获发酵剂（2,000 xg离心; 4℃; 10分钟）。用5毫升的无菌盐水的，洗细胞，然后重悬细胞沉淀。保持细胞在冰上。 </li></ol></li><li>制备（pH 7）中介质24琼脂平板含有25毫升的SH为1.5％（重量/体积）的琼脂。 </li><li>一旦琼脂凝固，散布50微升的5，50和500上的琼脂毫安排储备溶液分别获得0.01，0.1和1.0毫最终CEPA浓度。建立未处理和溶剂对照板，其中包括没有修改和50微升的溶剂的扩频用于溶解的测试化合物。 </li><li>为K的一个循环满（〜5微升）分离的菌落的Streak平板xylinus启动文化和石蜡膜密封。接种所有板块一式三份。在30℃下孵育板五天。 </li><li>辐透使用数字USB显微镜和质量在放大20倍ograph殖民地评估固体培养基上菌落形态和纤维素的生产。 注:纤维素表现为沿菌落边缘朦胧物质。 </li><li>为了控制氯和磷酸盐水平，执行使用0.01,0.1相同的实验，并用5，50和500毫米的股票，分别的NaCl的NaH 2 PO 4溶液的1.0mM的。 </li></ol> 5.薄皮测定<ol><li>成长和量化K. xylinus发酵剂培养物一式三份: <ol><li>一式三份，接种K的单菌落xylinus到5ml的SH培养基（pH为5），补充了0.2％（体积/体积）过滤灭菌纤维素酶。孵育在30℃下以150rpm搅拌培养物，直到OD达到0.3〜0.4 600（约72小时）。 </li><li>通过离心收获发酵剂（2,000 xg离心; 4℃; 10分钟）。用5毫升无菌盐水，冲洗Ť他细胞两次，重悬细胞沉淀。保持细胞在冰上。 </li><li>量化使用佩特罗夫，豪塞尔计数室细胞。 </li></ol></li><li>准备预混接种24孔板: <ol><li>补充60毫升的SH培养基（pH为7）与120微升的5，50和500毫CEPA股以获得0.01，0.1和1.0毫最终CEPA浓度分别。补充另60毫升SH培养基（pH为7）与120微升用于溶解CEPA溶剂的。旋涡混合。 </li><li>划分各60毫升含有CEPA培养基成4个14毫升分装。以10 5个细胞/ ml的浓度接种三连起子培养的生物学重复14毫升等分试样。保持与管细胞在冰上以防止纤维素的生产。 注:其余14毫升一份将用于无菌对照孔。 </li></ol></li><li>接种24孔板: <ol><li>测试在其自己的板各处理与生物学重复的三排，并无菌控件的行（ 图2）。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/54682/54682fig2.jpg" /> 图2:示出了用于防护薄膜组件测定和分析库存CEPA补充的pH为7 SH培养基（60毫升） 的协议流程图等分为三个独立的生物复制接种和无菌控制（各14ml）中。然后，这些培养物等分至六个技术重复（2ml）中于24孔板，然后用石蜡膜密封。在30℃下培养7天后，菌膜收获和特点是由FT-IR确定湿重，厚度，干重，和结晶度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig2large.jpg" target="_blank">请点击此处查看该图的放大版本</a> <ol start="2" style="margin-left: 40px;"><li>使用14毫升主结构，加2ml到EAC无菌24孔板六个孔小时。完成三个生物学重复和无菌控制（ 图2）。重复每个处理。 </li><li>用石蜡膜密封板，并在30℃下静态孵育7天。 </li></ol><ol start="4"><li>采收和测量薄皮湿重，厚度，干重（纤维素产量）和结晶度（ 图2）: <ol><li>压下薄膜的一侧提升对方薄膜边缘并移除个人药膜用钳子。在保持抓地力，把他们放在新鲜的纸巾在3秒内称重，以确定其湿重之前删除多余的网上平台。 </li><li>相邻对齐使用高分辨率数码相机从侧面一把尺子和照片药膜。 </li><li>使用ImageJ软件17，测量左肩，右肩各薄膜的薄膜中心厚度。平均所有技术复制每个生物replicat即</li><li>单独的菌膜转移到6孔板的孔中。在80℃下用12ml 0.1N NaOH中的治疗药膜20分钟以裂解细胞。 </li><li>取出NaOH和用超纯水清洗与搅拌24小时中和药膜。换水每隔6小时。 注意:药膜应该是洗涤步骤完成后白色。 </li><li>上硅垫地方药膜和在50℃下干燥48小时至恒重。干燥后，从垫取出，并测量薄膜上的权重分析的规模，以确定细菌纤维素产量。 </li><li>使用傅立叶变换红外光谱使用32次扫描（FT-IR）和4cm的分辨率-1 4000至650厘米范围内分析薄膜结晶-1。计算结晶度指数，CI（IR），采用了1437 / A 895;在"晶体乐队"和"非晶带"的吸收比前面所描述的18。 </li></OL&gt; </li><li>为了控制氯和磷酸盐水平，执行使用0.01,0.1相同的实验，并用5，50和500毫米的股票，分别的NaCl的NaH 2 PO 4溶液的1.0mM的。 </li><li>分析数据的Pellicle: <ol><li>通过确定薄膜湿重和干重之差计算防护膜水合。 </li><li>平均所有技术重复的值，以获得用于统计分析每个生物复制一个值。比较使用单向ANOVA和Tukey多重比较检验处理。差异显著如果p &lt;0.05。 </li><li>正常化的数据作为未处理的对照组的百分比和绘制生物学重复的平均值。 </li></ol></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+studies+of+the+thermal+decomposition+of+2-chloroethylphosphonic+acid+in+aqueous+solution.">Kinetic studies of the thermal decomposition of 2-chloroethylphosphonic acid in aqueous solution.</a> Plant Physiol. 58 (5), 700-702 (1976).</li><li>Klein, I., Lavee, S., Ben-Tal, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+water+vapor+pressure+on+the+thermal+decomposition+of+2-chloroethylphosphonic+acid.">Effect of water vapor pressure on the thermal decomposition of 2-chloroethylphosphonic acid.</a> Plant Physiol. 63 (3), 474-477 (1979).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio+assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio assays with tobacco tissue cultures.</a> Physiol. Plant. 15 (3), 473-497 (1962).</li><li>Schramm, M., Hestrin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+production+of+cellulose+at+the+air/liquid+interface+of+a+culture+of+Acetobacter+xylinum.">Factors affecting production of cellulose at the air/liquid interface of a culture of Acetobacter xylinum.</a> J. Gen. Microbiol. 11 (1), 123-129 (1954).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9 (7), 671-675 (2012).</li><li>Ciolacu, D., Ciolacu, F., Popa, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amorphous+cellulose-structure+and+characterization.">Amorphous cellulose-structure and characterization.</a> Cellul. Chem. Technol. 45 (1), 13-21 (2011).</li></ol>

The authors have nothing to disclose.

此处所描述的方法使用的模式生物，K勾勒出在从CEPA 就地产生乙烯的细菌乙烯反应的研究xylinus。这种方法是非常有用的乙烯可以通过补充有pH值大于3.5 10,11-用CEPA否定为纯乙烯气或专门的实验室设备的需要的任何含水介质来制备。这种方法不限定于学习CEPA衍生乙烯对细菌的效果，但也可以适用于研究在真核生物乙烯反应。重要的是平行的对照实验，用磷酸盐和氯化物，因为这些化合物也被从原位安排分解产生来执行。另一个必要的控制是确定安排本身是否施加在所研究的微生物培养物的直接作用。生理上不相关的乙烯浓度时产生K. xylinus我们的文化重新生长在pH值为5 SH媒介，因此，作为CEPA效应9控制;然而，这种方法只适用于酸耐受微生物。 在含水生长培养基的使用对生物研究CEPA衍生的乙烯需要验证，即乙烯确实正在系统内产生的。这里所描述的方法利用了A.拟南芥三重响应测定法，其利用由黑暗生长的苗中乙烯1的存在下表现出的三重响应表型。培养皿中分为四个象限让幼苗在MS培养基单独生长，但邻近于相关的微生物的生长培养基。 CEPA的解决方案到微生物生长培养基应用程序方便乙烯生产，并公开相邻苗乙烯外源，因为它渗入顶空。补充与ACC的MS培养基提供了一个积极的控制，因为它增强内源性产品由A.乙烯离子拟南芥和三重反应表型诱导以后。 培养液pH值是另一个重要的考虑因素从CEPA中微生物的生长培养基13,14生产乙烯的时候。通常用于培养K的SH培养基xylinus的pH为约5基本上降低了生产安排衍生乙烯（Augimeri和表带，未发表的数据）;因此，将培养基的pH值调至7 9，这与其它研究，证明了安排击穿的速率处于或低于pH 5极其缓慢，而这是在pH 7 13大大增强是一致的。以确保因此，必须该测试生物体的生长培养基保持高于pH 5 K. xylinus培养物达到约5.5的最低pH值，使乙烯9的演变。要注意的是乙烯的绝对浓度不能根据这些培养c是控制是很重要的onditions。 CEPA衍生乙烯上生长，纤维素生产，和K的薄膜性能的影响xylinus用于说明细菌乙烯的反应可以用安排检查。适应这些方法来研究在其他生物体乙烯的反应是在更换生长培养基和表型分析一样简单。利用CEPA的为研究人员提供与乙烯气体方便的替代品，可能会鼓励更多的细菌乙烯介导的研究。

烯烃乙烯（C 2 H 4）首次被发现在1901年的植物激素被观察到，豌豆苗，在使用煤气灯一个实验室中生长，显示出不正常的形态，其中茎（胚轴）为较短时，较厚的和弯曲相比普通豌豆苗横盘整理;表型后称为三重反应1,2。随后的研究表明，乙烯是一种调节多种发育过程如生长，应激反应，果实成熟衰老3。 拟南芥植物生物学研究模式生物的重要激素，已在关于其对乙烯反应得到很好的研究。几个乙烯反应突变体已经被分离通过利用在黑暗生长的答中观察到的三联反应表型拟南芥秧苗在乙烯1,4,5的存在。生物合成的前体为乙烯生产厂是1-一个minocyclopropane羧酸（ACC）6和三重反应测定期间通常用于增加内源性乙烯产量，导致三联响应表型1,4,5。 虽然乙烯反应被广泛研究在植物中，外源乙烯对细菌的效果大大充分研究，尽管细菌与植物的密切联系。一项研究报告指出某些假单胞菌株可以使用乙烯作为碳源和能源7的唯一来源生存。然而，只有两个研究已经证明，细菌乙烯反应。第一项研究表明， 铜绿假单胞菌 ，P的菌株荧光假单胞菌 ，P.恶臭 ，和P.丁香使用琼脂糖塞测定，其中熔融的琼脂糖用纯乙烯气8平衡的趋化性缓冲液混合分别朝向乙烯趋化。但是，据我们所知，没有出现过菲尔特采用纯乙烯气体呃报告表征细菌乙烯反应，可能是由于处理实验室气体没有专业设备的困难。细菌乙烯反应的第二份报告表明，乙烯增加细菌纤维素生产的影响基因表达的水果相关的细菌，Komagataeibacter（原Gluconacetobacter）xylinus 9。在这种情况下，乙烯-释放化合物，2-氯乙基膦酸（CEPA）用于细菌生长介质内就地产生乙烯，绕过用于纯乙烯气体或专门的设备的需要。 - 14通过一碱催化的，一级反应12 1的摩尔比高于pH 3.5 10,11:CEPA在1产生乙烯。 CEPA的降解在生产乙酯与pH和温度13,14和结果正相关烯，氯和磷酸盐。 CEPA提供兴趣研究细菌反应，乙烯与方便的替代乙烯气体的研究。 以下协议的总体目标是提供一种简单而有效的方法来研究细菌乙烯反应，并且包括从安排分解细菌生长培养基中生产乙烯的生理相关水平的验证，培养物pH的分析，以确保CEPA分解期间不受损细菌生长，和的乙烯对细菌形态和表型的影响的评估。我们演示使用K.这些协议xylinus，然而，这些协议可以适于通过使用适当的生长培养基中和表型分析，以研究在其它细菌乙烯反应。

<table border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">1-aminocyclopropane carboxylic acid (ACC)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">A3903</td>
			<td style="width:432px;">Biosynthetic precursor of ethylene in plants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">4-sector Petri dish</td>
			<td style="width:136px;">Phoenix Biomedical</td>
			<td style="width:119px;">CA73370-022</td>
			<td>For testing triple response</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Agar</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">AGR001.1</td>
			<td>To solidify medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Canon Rebel T1i DLSR camera</td>
			<td style="width:136px;">Canon</td>
			<td style="width:119px;">3818B004</td>
			<td>For pictures of pellicles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Cellulase from Trichoderma reesei ATCC 26921&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C2730</td>
			<td>Aqueous solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Citric acid</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">CIT002.500</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Commercial bleach</td>
			<td style="width:136px;">Life Brand</td>
			<td style="width:119px;">57800861874</td>
			<td>Bleach for seed sterilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Concentrated HCl</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">HCL666.500</td>
			<td>Hydrochloric acid for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital USB microscope</td>
			<td style="width:136px;">Plugable</td>
			<td style="width:119px;">N/A</td>
			<td>For pictures of colonies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ethephon (&#8805; 96%; 2-chloroethylphosphonic acid)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C0143</td>
			<td>Ethylene-releasing compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glucose</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">GB0219</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Komagataeibacter xylinus ATCC 53582</td>
			<td style="width:136px;">ATCC</td>
			<td style="width:119px;">53582</td>
			<td>Bacterial cellulose-producing alphaproteobacterium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microcentrifuge tube</td>
			<td style="width:136px;">LifeGene</td>
			<td style="width:119px;">LMCT1.7B</td>
			<td>1.7 mL microcentrifuge tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Murashige and Skoog (MS) basal medium&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">M5519</td>
			<td>Arabidopsis thaliana growth medium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Na2HPO4&#183;7H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPD579.500</td>
			<td>Sodium phosphate, dibasic heptahydrate for SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaCl</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">SOD001.1</td>
			<td>Sodium chloride for saline and control solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">NaH2PO4&#183;H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPM306.500</td>
			<td>Sodium phosphate, monobasic monohydrate for control solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaOH</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SHY700.500</td>
			<td>Sodium hydroxide for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Paraffin film</td>
			<td style="width:136px;">Parafilm</td>
			<td style="width:119px;">PM996</td>
			<td>For sealing plates and flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Peptone (bacteriological)</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">PEP403.1</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Petroff-Hausser counting chamber</td>
			<td style="width:136px;">Hausser scientific</td>
			<td style="width:119px;">3900</td>
			<td>Bacterial cell counting chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Polyethersulfone sterilization filter 0.2 &#181;m</td>
			<td style="width:136px;">VWR</td>
			<td style="width:119px;">28145-501</td>
			<td>For sterilizing cellulase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sucrose</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SUC600.1</td>
			<td>Sucrose for MS medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Yeast extract</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">G0961</td>
			<td>For SH medium</td>
		</tr>
	</tbody>
</table>

utilizing the ethylene-releasing compound, 2-chloroethylphosphonic acid, as a tool to study ethylene response in bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

示意图板设置从CEPA乙烯解放SH培养基（pH值为7）经三重反应实验验证如图1A - ℃。示出了防护薄膜组件的协议的流程图在图2中黑暗生长的A中示出拟南芥幼苗展现的三重反应表型（以一种夸张的顶钩较短和较厚的下胚轴）中ACC的存在下，并在通过CEPA的SH培养基上分解产生乙烯（pH 7）中的存在，但未经处理的条件下，不 （ 图3A）9。 ACC-和安排衍生乙烯处理A的下胚轴长度拟南芥种子在显著（P &lt;0.0001）比未处理的对照组（ 图3B）9，确认乙烯在生理相关concentra从安排被释放的SH培养基（pH为7）上短化。未处理和安排处理的K的pH值xylinus文化仍高于5（ 图4）9;因此CEPA分解成乙烯不损害由于细菌有机酸的生产。 CEPA衍生的乙烯增加细菌纤维素生产时K. xylinus生长固体的SH培养基（pH 7）中 （ 图5）9。 CEPA衍生乙烯所有浓度显著降低防护膜湿重（ 图6A），并没有影响薄膜厚度（ 图6B）和显著增加防护膜的干重（纤维素收率; 图6C）9。采取防护薄膜厚度测量的代表性照片示于图7。菌斑水化减少了安排衍生乙烯（ 图8A）9的所有浓度，而安排衍生所有浓度乙烯9增加像素licle结晶度（ 图8B）。氯化钠和NaH 2 PO 4的影响是在所有情况下显着（数据未显示）证实观察到的表型是由安排衍生乙烯引起的。 <img alt="图3" src="/files/ftp_upload/54682/54682fig3.jpg" /> 图3:CEPA 分解SH培养基（pH值7）生产乙烯数码USB显微镜照片显示黑暗生长A.。拟南芥秧苗显示三重反应型（短下胚轴，厚的下胚轴和夸张的顶钩）相比未处理（A）在ACC和CEPA衍生的乙烯的情况下生长时。在ACC和安排衍生乙烯的存在下生长的幼苗的下胚轴长度分别significantlý比未处理的对照（B）的短。比例尺= 1毫米。误差线显示标准差（n = 3）。用不同的字母酒吧显著差异（p &lt;0.0001）。这个数字已经从Augimeri和带9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54682/54682fig4.jpg" /> 图4:K 的pH值xylinus培养仍然是SH培养基（pH 7）中。将培养的pH保持5以上，允许安排的高效分解成乙烯整个细菌生长周期安排分解不够高 。 K的pH值因为它们分泌xylinus培养必须监测和再吸收有机酸。图例显示测试CEPA的浓度。需要注意的是y轴开始在pH 5误差线显示标准差（n = 3）。这个数字已经从Augimeri和带9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54682/54682fig5.jpg" /> 图5:CEPA 衍生的乙烯由K.增加纤维素生产一个固体培养基上xylinus。K.或CEPA（F - H -用酸化（pH 2.5）的超纯水（B）中，磷酸盐和氯化物（E C）xylinus生长上是未处理的（A）中 ，或SH琼脂板（pH 7）中预处理）。代表性的菌落被示出。由K.生产的箭头所示纤维素xylinus，视为菌落周围朦胧的物质。比例尺= 0.5mm左右。这个数字公顷Ş从Augimeri和带9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54682/54682fig6.jpg" /> 图6:CEPA 衍生乙烯降低保水能力，并增加K的产率xylinus纤维素药膜。培养物在24孔板补充有CEPA SH培养液（pH 7）中静态培养，并在30℃下培养7天，收获和分析之前药膜。 CEPA衍生乙烯减小防护膜湿重（A）中，对防护薄膜组件的厚度（B）和增加的防护膜干重（C）的任何影响。不同的安排处理为不彼此显著不同。误差线显示标准差（n = 3）。与酒吧不同字母的显著差异（p &lt;0.05）。这个数字已经从Augimeri和带9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig6large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54682/54682fig7.jpg" /> 图7:K的代表照片xylinus药膜。防护薄膜厚度由使用ImageJ软件照片来测量。比例尺= 10毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54682/54682fig8.jpg" /> 图8:CEPA 衍生降低乙烯的水合通过增加防护膜的结晶度K. xylinus纤维素药膜。培养物在24孔板中的SH培养液（pH 7）中静态培养，并在30℃下培养7天，收获和分析之前药膜。防护薄膜组件水合计算为防护膜湿重和干重之差。通过增加防护膜的结晶（B）的安排衍生乙烯减少防护薄膜水化（A）中。不同的安排处理为不彼此显著不同。误差线显示标准差（n = 3）。用不同的字母酒吧显著差异（p &lt;0.05）。这个数字已经从Augimeri和带9修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54682/54682fig8large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

Watch this Scientific Journal Video about Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria at JoVE.com

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

utilizing-ethylene-releasing-compound-2-chloroethylphosphonic-acid-as

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.8K 次观看.  University of Ontario Institute of Technology. The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas. 

视频: Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of &#945;2&#946;1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

A detailed protocol to perform a titration ELISA is described. Moreover, a novel algorithm is presented to evaluate titration ELISAs and to obtain a dissociation constant of binding of a soluble ligand to a microtiter plate-immobilized receptor.

滴定 ELISA 法测定受体配体相互作用的离解常数

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

从鸡蛋黄中分离脂蛋白颗粒,研究细菌病原体脂肪酸并入膜磷脂

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

在Vivo羟基基蛋白足迹研究卡诺哈布迪特人的蛋白质相互作用

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

利用时间分辨蛋白诱导的荧光增强来鉴定稳定的局部构象，一次一α-突触核蛋白单体

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

用于研究真核糖原代谢的分光光度法

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...