这是非常重要的，穿在编制了一层薄薄的无粉手套。 清洁样板<ol><li>使用不锈钢或黄金的MALDI样品板。 </li><li>用甲醇清洗和Kimwipe轻轻擦拭。不要揉搓或刷洗Kimwipe的表面，防止刮伤表面。 </li><li>洗净与H 2 O和Kimwipe轻轻擦拭。 </li><li>用甲醇清洗和Kimwipe轻轻擦拭。 </li><li>如果需要，重复甲醇/ H 2 O /甲醇清洗周期，总是与甲醇结束。 </li><li>确保有没有鬼点或盘上的剩余残渣。如果有鬼魂点，与去离子水冲洗，而摩擦表面用戴手套的手（不要擦Kimwipes）板。重复甲醇/ H 2 O /甲醇清洗周期。 </li><li>使用后立即清洗板。 </li></ol>一层薄薄的底物溶液的制备<ol><li>混合1饱和4 HCCA（乙腈2部分，1份水，0.1％TFA的最终）和3部分异丙醇。 注：薄层基板的解决方案是非常稳定，可在室温下保存在黑暗的一年。这也可能是保存在4 ° C。 </li></ol>制作薄层基板<ol><li>于薄层板左侧的中心，并与一个枪头一侧蔓延的底物溶液20 -50μL。扫一个方向。 </li><li>随着解决方案的干燥，有机溶剂的蒸发速度非常快，离开板面背后分钟的水液滴。在这一点上，包裹层的Kimwipe最初轻轻吸干板表面与你的食指。 </li><li>当表面上是从水滴中解脱出来，擦拭整个板块使用Kimwipe单向席卷议案。 注：如果解决方案不扩散到整个板面不够，增加乙腈组成和重新薄层。一些参数可能影响传播的解决方案，包括环境温度和湿度。 注：与黄金板块，该层通常会出现一种黄色的反思，查看和光线角度而定。用钢板，只有边缘层通常是可见的。 </li><li>清洁与新鲜Kimwipe板的边缘，然后放置在盘子持有人，以防止污染仪器矩阵。 </li></ol>基质溶液的制备<ol><li> <a href="http://prowl.rockefeller.edu/protocols/saturated-4HCCA-solution.html">饱和4 HCCA</a>在FWI（甲酸3个部分，1份水，2份异丙醇）膜和可溶性蛋白的分析建议。它也可用于在特殊情况下的分析非常疏水的大肽（M&gt; 4000 U）。确保矩阵的解决方案，用于发现的蛋白质不超过〜50％的有机质含量，因为它会完全溶解的薄层基板，击败了该方法的优势。 </li></ol>薄层基板测试<ol><li>盘上的矩阵解现货0.5μL。发白的沉淀物，将出现在薄薄的一层和液滴之间的接口。当这层完全涵盖液滴底部，通常在10-15秒之内，吸出多余的液体真空线。如果需要超过30秒的形式沉淀，这是一个迹象表明，可能有一层薄薄的底物和/或矩阵解的问题。 </li></ol>示例应用程序<ol><li>在开始解决方案的分析物的浓度应在10和1000μM。 </li><li>在矩阵解到最后一个介于0.2和2μm的分析物的浓度稀释的待测溶液。例如，开始加入​​1μl样品和基质溶液9μl。 注意：请确保该矩阵是接近饱和的最终解决，否则你将溶解的薄层基板。 注：获得体面的信号所需的分析物的浓度是高度依赖的解决方案的复杂性和成分（例如，污染物）和分析物的电离度。 注：如果单步稀释，不会产生良好的效果，你应该尝试另一种稀释步骤。虽然反直观的，高度集中的分析物的解决方案很少产生良好的光谱。 注 ：膜蛋白通常需要较大的稀释。 </li><li>现货样品/基质混合物0.5μL板。发白的沉淀物会形成薄薄的一层和液滴之间的接口。这种沉淀是基质和分析物cocrystallization。当这层完全涵盖液滴底部，乌苏市在10-15秒内的盟友，吸真空线多余的液体。如果需要长于30秒的形式沉淀，它可能是一种迹象的污染物目前，以防止结晶，或您的分析物过于集中。 </li></ol>校准应用程序<ol><li>准备以类似的方式，最后的校准浓度在0.1 -0.5μm的之间的校准/基体的混合物。 </li><li>现货0.5μL校准/基体的混合物，在接近样本点板。这个物理接近伪内部校准程序，在该板块是从样本点转移到样品采集期间的现场校准，添加几个校准物的激光脉冲的频谱使用。这种方法可以保证较高的质量只比外部校准精度的校准。 </li></ol>洗涤步骤<ol><li>大约一个冰冷的0.1％的水溶液TFA的解决方案2μL清洗每一个部位。多余的液体吸真空线。 </li></ol>收购质谱<ol><li>您现在已经准备好收购质谱。如果您使用的FWI矩阵的解决方案，您必须获得您的光谱尽快甲酰如待测防止不可逆转的修改。 </li></ol>

<ol>
	<li>Xiang, F., Beavis, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+to+Increase+Contaminant+Tolerance+in+Protein+Matrix-Assisted+Laser+Desorption+Ionization+by+the+Fabrication+of+Thin+Protein-Doped+Polycrystalline+Films.">A Method to Increase Contaminant Tolerance in Protein Matrix-Assisted Laser Desorption Ionization by the Fabrication of Thin Protein-Doped Polycrystalline Films.</a> Rapid Communications in Mass Spectrometry. 8, 199-204 (1994).</li><li>Cadene, M., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Robust,+Detergent-Friendly+Method+for+Mass+Spectrometric+Analysis+of+Integral+Membrane+Proteins.">A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins.</a> Analytical Chemistry. 72, 5655-5658 (2000).</li><li>Hook, P., Mikami, A., Shafer, B., Chait, B. T., Rosenfeld, S. S., Vallee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+allosteric+control+of+cytoplasmic+dynein+ATPase+activity+by+the+stalk+and+C-terminal+domains.">Long-range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains.</a> J Biol Chem. 280, 33045-33054 (2005).</li></ol>

至关重要的是，所有试剂的最高质量（如HPLC级）。 无粉手套（乳胶或丁腈） 不锈钢或黄金的MALDI样品板 甲醇（MeOH） 水 Kimwipes α-氰基-4 - 羟基苯甲酸（4 - HCCA） 三氟乙酸（TFA）（终浓度为0.1％H 2 O） 乙腈（ACN） 甲酸 异丙醇

maldi sample preparation: the ultra thin layer method

本视频演示了矩阵/分析物的分析肽和蛋白质基质辅助激光解吸电离质谱（MALDI - MS的）1，2层超薄的准备。超薄层的方法，涉及生产样板，作为一个矩阵/分析物的混合物随后结晶播种地面基材层的基质晶体（α-氰基-4 - 羟基苯甲酸）。比其他样本沉积的方法（如干燥液滴）的超薄层的方法的优点是，它提供了（我）更大的宽容，杂质，如盐和洗涤剂，（二）更好的分辨率，（三）更高的空间统一。精确质量测定的蛋白质，这种方法尤其有用。该协议最初是开发和优化膜蛋白的分析，并成功地用于分析离子通道，代谢产物的运输，和受体，含有2和12个跨膜结构域2之间。由于原来的出版物，它也显示出可溶性蛋白的分析同样有用。事实上，我们已经使用了大量的蛋白质的属性的范围很广，包括那些具有高的分子量为380 kDa的3。这是目前我们所有的蛋白质分子质量分析方法的选择。所描述的过程，坚持生产高品质的光谱，和它是敏感的，功能强大，且易于实现。

Watch this Scientific Journal Video about MALDI Sample Preparation: the Ultra Thin Layer Method at JoVE.com

MALDI Sample Preparation: the Ultra Thin Layer Method

这个视频演示的超薄层肽和蛋白质基质辅助激光解吸电离质谱（MALDI - MS的）分析的矩阵/待测的准备。

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 ...

maldi-sample-preparation-the-ultra-thin-layer-method

Research

JoVE Journal

Biology

19.2K 次观看.  Rockefeller University. 这个视频演示的超薄层肽和蛋白质基质辅助激光解吸电离质谱（MALDI - MS的）分析的矩阵/待测的准备。

视频: MALDI Sample Preparation: the Ultra Thin Layer Method

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

科研

生物学

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

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

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

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

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 ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

样品制备质谱术分析

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

使用微蒂特萝卜无线电标签进行多维欧大肠杆菌测量 (p) ppGpp,然后是薄层色谱

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...