这项工作是由美国国家科学基金会克里斯托夫本宁堡的赠款支持。

1。脂质提取<ol><li>开始收获30毫克，从4周龄琼脂上生长的植物拟南芥叶片凝固介质或土壤和他们转移到1.5 mL聚丙烯反应管脂质提取。鲜叶可闪光冷冻在液氮中，并储存在-80 ° C </li><li> ，氯仿和甲酸（20点10分01秒，V / V / V），每个样品中加入300μL提取溶剂甲醇组成。用力振摇5分钟（使用的油漆摇床或类似）。 </li><li>加入150μL0.2 M磷酸（H 3 PO 4），1米氯化钾（KCl）和涡简要。 </li><li> 13000 XG在室温离心1分钟。溶解在氯仿逐步降低血脂，将会发现到薄层板。 </li></ol> 2。薄层色谱法（TLC）15 <ol><li>要准备薄层板，淹没装载30秒到0.15 M硫酸铵（（NH 4）2 SO 4）解决方案的地带一个20cmx20cm硅胶涂薄层板上，淹没了30秒后，干板至少2天有盖容器内。在激活过程中铵的升华留下硫酸，质子化磷脂必要从其他甘油脂分离。 </li><li>在一天的实验，激活薄层色谱板在烤箱烘烤在120 ° C为2.5小时。 </li><li>激活板室温冷却后，用铅笔画出一条直线从板边（1.5厘米）的色谱的起源整个板块。 </li><li>在通风橱中，慢慢地提供3 + X在较低的氯仿阶段使用200μL的黄色塑料提示下慢流的 N 2 20微升移液器20μL脂质提取物。为此，聚乙烯管是连接的N 2坦克的监管。保持现货直径大于1厘米的小。每个板最多可容纳10个样品（随后GLC分析计划时）。 </li><li>由于脂质点通风柜完全干燥，准备发展溶剂甲苯，丙酮，水（91毫升:30毫升:7.5毫升）组成。如果周围的空气相对湿度高，可能会受到影响分离。水在这种情况下，应减少（91毫升:30毫升:7.0毫升），以达到理想的分离。 </li><li>倒入80毫升，发展成一个密封室（L:高:宽= 27.0:26.5:7.0厘米/厘米/厘米）的薄层色谱溶剂与样品结束朝下放入罐板。使用钳密封罐。溶剂，将提升该板块将被分离和血脂。开发时间是在室温下约50分钟。 </li><li>当溶剂的前锋已经达到了1厘米板顶部，小心取出从水箱板，并完全干燥约10分钟的通风柜。 </li><li>薄层色谱法分离血脂可与碘进行定量分析或不可逆转地用硫酸或α-萘酚染色简要可逆染色。 <ol><li>硫酸炭化:喷以50％硫酸在水中15分钟（图2A）在通风柜和烘烤在120℃的玻璃喷雾瓶板。 </li><li> α-萘酚糖脂染色:在120 ° C的板喷2.4％（W / V）10％α-萘酚（V / V）硫酸，80％（V / V）乙醇和烘烤3-5直到糖脂乐队分钟被染成粉红色或紫色（图2B）。过度治疗会导致血​​脂，由于存在硫酸试剂炭化。 </li><li>碘染色:在通风橱中，放入一个封闭的薄层色谱与碘晶体罐（在一个托盘上领先的碘蒸气饱和的气氛，直到血脂是可见的底部）的板。不要暴露板太长碘碘共价键修改多不饱和脂肪酸（图2C）。另外，要避免任何氧化脂质，穿插样品车道的唯一标准线应使用巴斯德吸管插入碘晶体通过二氮是对个别标准泳道吹玻璃羊毛染色。 </li></ol></li></ol> 3。脂肪酰基酸甲酯（FAME），反应16 <ol><li>从薄层板上，用刀片取出硅周围确定的脂斑。刮脂含二氧化硅和转让硅微粉，玻璃管与聚四氟乙烯（PTFE）内衬聚四氟乙烯螺丝帽使用一个漏斗。 </li><li>添加无水甲醇1毫升1N盐酸（盐酸）每个样品由玻璃吸管。 </li><li>加入100微升50微克毫升-1十五烷酸（15:0）作为内部使用200μL200μL的黄色塑料尖吸管标准每个样品200微升移液器。 pentadecenoic甲醇盐酸酸作为控制，保持管。紧紧关闭的玻璃管与聚四氟乙烯内衬的盖子。 </li><li>孵育GL屁股管在80℃水浴25分钟。管需要密封，使溶剂不蒸发。 </li><li>管冷却下来后，加入1毫升0.9％氯化钠1 mL正己烷和涡大力。在1000xg离心3分钟的样品。 </li><li>在通风柜中，删除与巴斯德吸管样品的正己烷/上层，并放置到一个新的13x100毫米玻璃管。 </li><li>完全未经干燥的正己烷蒸发下慢流的 N 2 。 </li><li>在60μL正己烷溶解，导致脂肪酰基methylesters小号。转移到自动进样瓶和盖紧紧样本。样品可存放在4 ° C的短期和-20℃的几天。 </li></ol> 4。气相色谱（GLC）10 <ol><li>在开始GLC之前，确保充满氦气，氢气和气缸。 </li><li>必须补充足够的正己烷溶剂水库和废物的容器必须是空的。对于脂肪酰基methylesters分离，附加到本机的DB - 23列。 </li><li>到自动进样器瓶。启动绿灯ChemStation软件系统的计算机上。 </li><li>入口温度设定在250 °与氦流量48.6毫升分钟-1和21.93 PSI的压力分流比为30.0:1。 </li><li>最初在140 ° C烘箱温度设置为2分钟，并提升至160 ° C在25℃ 分钟 -1率。然后设定温度增加160 ° C到250 ° C时，利率为8 ° C最低-1，并保持在250 ° C为4分钟减少至140 ° C率在38 ° C 最低 - 1。一个运行大约需要21分钟。 </li><li>的火焰离子化检测器温度为270 ° 30.0毫升分钟 -1，400毫升分钟 -1和30.0毫升分钟 -1氦流量的空气流速与氢气流量</li><li>输入小瓶和运行序列表中的样本名。将10μL的注射器，注入2％μL样品小瓶。 </li><li>当仪器已准备就绪，开始运行顺序。 </li></ol> 5。代表性的成果: 从4周龄的拟南芥幼苗薄层色谱分离血脂不可逆转染色的例子如图2所示。硫酸染色血脂（图2A）被烧焦和褐色斑点出现。是首选α-萘酚染色，如MGDG糖脂，DGDG，SQDG与α-萘酚进行一个粉红色的紫色，而其他的极性脂类染色黄色（图2B）染色等糖脂。碘染色是可逆的，并给出血脂一个黄色超过碘蒸发（图2C）短的时间内即会消失。简要碘染​​色血脂可以受到GLC分析，虽然未染色血脂是可取的，以减少分解脂类。 如果成功，将观察到的鲜明信号，代表不同的脂肪酰基甲酯后绿灯（图3）。脂肪酸碳链较短和较少的双键酰基甲酯的保留时间较短，使用DB - 23列。脂肪酰基甲酯分析，找出与脂质成分改变的突变体是一个敏感的工具。在图4中，MGDG18:3脂肪酸的摩尔比是下降tgd4 - 1突变体比野生型的18。除以所有脂质类的痣类脂质的脂肪酰基甲酯的痣，每个脂质的摩尔比计算。例如，要计算的MGDG的摩尔比: （MGDG）MOL％=Σ[脂肪酸甲酯（MGDG）] /Σ[脂肪酸甲酯（总）] X100％ 从野生型和突变导致摩尔比率每个脂质类可以比拟的。例如，tgd4 - 1突变体增加了MGDG和PG的相对含量，但减少DGDG和PE（ 图 5） 18的金额。 <img src="/files/ftp_upload/2518/2518fig1.jpg" alt="图1" /> 图1。极性脂使用拟南芥幼苗的分析流程图。总脂是从4周龄的拟南芥幼苗中提取和薄层色谱法分离。 GLC分析酯交换板薄层色谱分离血脂可刮掉。 <img src="/files/ftp_upload/2518/2518fig2.jpg" alt="图2" /> 图2。血脂薄层板分离。野生型幼苗脂质提取物35毫克（鲜重）是分开的薄层色谱法和硫酸（一），α-萘酚（B）或碘蒸气（三）染色。三重复显示在每个染色法。 DGDG，digalactosyldiacylglycerol; MGDG，monogalactosyldiacylglycerol，PC，磷脂，PE，磷脂，PG，phosphatidylglyce列伊; PI，磷脂酰肌醇; SQDG，sulfoquinovosyldiacylglycerol。 <img src="/files/ftp_upload/2518/2518fig3.jpg" alt="图3" /> 图3。绿灯脂肪酸Methylesters分析（脂肪酸甲酯）来自野生型MGDG。脂肪酸甲酯是一个30米的毛细管柱分离，火焰离子化检测。十五烷酸（15:0）作为内部标准。 <img src="/files/ftp_upload/2518/2518fig4.jpg" alt="图4" /> 图4。COL2野生型（白色列）和tgd4 - 1突变体（黑列）MGDG脂肪酸。脂肪酸的碳双键的数目。三重复的平均值和标准偏差。 <img src="/files/ftp_upload/2518/2518fig5.jpg" alt="图5" /> 图5。极性脂组成的野生型COL2（白色列）和tgd4 - 1突变体（黑列）。 3个重复的平均值和标准偏差错误栏显示。

<ol>
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TLC与GLC耦合提供了一个稳健，快速的定量分析植物中的极性脂的工具。脂质成分的微小变化可以识别，因此，这种方法已被用于极性脂代谢途径1,20受损的突变体的大规模筛选。这种方法也被广泛用于监察为底物，利用极性脂的酶的活动。2,6,7 除了叶，根和种子或叶绿体和线粒体等亚细胞组分，如其他植物组织的脂质成分也可以以同样的方式确定。 这里使用的溶剂体系（丙酮，甲苯，水）是在植物中的糖脂和磷脂的分离进行了优化。然而，在tgd1，2,3,4突变体和孤立的叶绿体，TGDG运行与PE而tetragalactosyldiacylglycerol与PC机上运行。在这种情况下与氯仿，甲醇，醋酸和水（85:20:10:4，V / V / V / V）的溶剂系统是使用13 。有时二维薄层色谱法，使用两个不同的溶剂体系进行进一步分离糖脂和磷脂19。此外，在植物组织中，可直接由绿灯未经薄层色谱法5初步分离，以确定总脂肪酸的名誉反应。除了 ​​表现出的TLC - GLC系统，血脂分析中使用的另一种方法是基于直接电喷雾串联质谱 17 。在此方法中的初始色谱分离提取脂质被省略了。然而，这种方法需要昂贵的设备和经验丰富的人员，这使得常规分析在实验室或突变体筛选有用。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)

生物膜从不同的细胞环境。从一个单细胞到多细胞植物和动物，甘油脂，如卵磷脂或磷脂，形成双分子膜的化学细胞及其周围环境之间的交流的边界和接口，。植物细胞与动物不同的是，有一个特殊的光合作用的细胞器，叶绿体。错综复杂的叶绿体膜系统包含独特的甘油脂，即糖脂缺乏磷:monogalactosyldiacylglycerol（MGDG），digalactosyldiacylglycerol（DGDG），sulfoquinovosyldiacylglycerol（SQDG）4 。这些脂质的作用不仅仅是结构。在光系统I和II表示甘油脂在参与光合作用8,11的晶体结构，发现这些糖脂和其他甘油脂。在磷饥饿，DGDG转移到extraplastidic细胞膜，以弥补磷脂 9,12的损失。 我们这些脂质的合成和功能知识大部分来自已经组合的遗传和生化研究拟南芥 14。在这些研究中，一个简单的程序，为极地血脂分析是必不可少的脂质突变体的筛选和分析，将详细概述。叶脂质提取物薄层色谱（TLC）分离和甘油脂是可逆沾有碘蒸气。单体脂类刮薄层板上，并转化为脂肪酰基methylesters（脂肪酸甲酯），再加上火焰离子化检测器（FID - GLC）（图1）的气相色谱分析。该方法已被证明是一个可靠的工具，突变体筛选。例如，在tgd1，2,3,4内质网到质的脂质贩运突变体发现的基础上积累的异常galactoglycerolipid:trigalactosyldiacylglycerol（TGDG）和18:3的相对量减少（碳:双债券）在膜脂的3,13,18,20脂肪酰基群体。这种方法也适用于确定使用基板 6脂类蛋白质的酶活动。

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

极性脂提取物的成分和个别甘油脂脂肪酸组成在一个简单而强大的脂质分析实验确定。为此，甘油脂是孤立的薄层色谱法和酰基组transmethylation。气相色谱定量脂肪酰基methylesters。

拟南芥极地甘油脂分析薄层色谱（TLC）再加上气相色谱（GLC）

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

arabidopsis-thaliana-polar-glycerolipid-profiling-thin-layer

Research

JoVE Journal

Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

36.6K 次观看.  Michigan State University. 极性脂提取物的成分和个别甘油脂脂肪酸组成在一个简单而强大的脂质分析实验确定。为此，甘油脂是孤立的薄层色谱法和酰基组transmethylation。气相色谱定量脂肪酰基methylesters。

视频: Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

自动疏水层析蛋白质纯化用柱选择

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...