Name: a colorimetric method for measuring iron content in plants
Uploaded: 2018-09-07T13:30:00
Description: Watch this Scientific Journal Video about A Colorimetric Method for Measuring Iron Content in Plants at JoVE.com

この作品は、イスラエル農業 (#16-16-0003) 省の主任研究員からの助成金によってイスラエル省、科学、技術およびスペースによって支えられました。

1. 植物材料と成長条件
<ol><li>5 cm × 5 cm ポット当たり種子 1 つタバコ (品種サムスン) は標準的なポットの中に満ちています。トレイにポットを置きます。23 ° C の一定温度で長い一日 (16/8 h/暗) 条件の下で成長部屋の植物を育てるポットから排水まで水道水で灌漑します。</li>
	<li>50±5 日後、実験用に最適の濃度によると、灌漑で Fe 治療を開始します。たとえば、0 から 6 mm、可溶性鉄キレー?...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygenation+of+ferrous+iron.">Oxygenation of ferrous iron.</a> Industrial &amp; Engineering Chemistry. 53 (2), 143-146 (1961).</li><li>Jones, A. M., Griffin, P. J., Collins, R. N., Waite, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferrous+iron+oxidation+under+acidic+conditions+-+The+effect+of+ferric+oxide+surfaces.">Ferrous iron oxidation under acidic conditions - The effect of ferric oxide surfaces.</a> Geochimica et Cosmochimica Acta. 145, 1-12 (2014).</li><li>Hawkesworth, C. J., Kemp, A. I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+continental+crust.">Evolution of the continental crust.</a> Nature. 443 (7113), (2006).</li><li>Thompson, L. M., Louis, M., Troeh, F. R., Thompson, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soils and soil fertility. , (1973).</li><li>Krueger, B. J., Grassian, V. H., Cowin, J. 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植物組織の鉄の測定は、灌漑や他の環境条件の影響を評価するために非常に重要です。ここでは、他の植物種や組織に容易に合わせることができるタバコ葉における Fe コンテンツ測定のための簡単で正確な比色定量法について述べる。
比色法のための条件を最適化する、鉄の溶解性を許可するように低 pH 媒体 (pH < 1.0) を使用しました。書き込み処理を行った鉄のすべて...

鉄 (Fe) は、すべての生物に重要な微量栄養素です。植物の呼吸、光合成とクロロフィル生合成などの基本的なプロセスに関与のため必須微量栄養素1についてです。無料鉄多価イオンの高集積植物細胞酸化ストレスを引き起こすフリーラジカルのリリースにつながる反応のために有害です。植物細胞内鉄の恒常性を維持するために、イオンは液胞に格納され、フェリチン、鉄の恒常性2に直接関与するタンパク質ケージおよびすべての生きている有機体の鉄の主要な記憶域構造内で隔離。同時には、鉄欠乏性貧血は、植物鉄 biofortification の増加が必要で、その結果、人間の人口のかなりの割合を影響します。植物フェリチンのユニークな性質上、フェリチン鉄食品濃縮栄養失調3のこの問題を戦うために有望な戦略を提供しています。
鉄イオンは、主に 2 つ酸化状態、すなわち鉄 (2価 Fe2 +または鉄 (II))、鉄 (三価 Fe3 +または鉄 (III)) フォーム。鉄、鉄クラスター4などのいくつかの他の形態はまた細胞であります。Fe はセルと自然フォーム ヘマタイト (Fe2O3) と ferryhidrites 内の酸化鉄として格納されます ((Fe3 +)2O3•0.5 H2O)5生理学的な条件の下で。これらの反応は、特に鉄のフォームに形成された水酸化物非常に低い容解性があります。鉄保持は、pH 56上固体状態で主はその結果、溶液の pH によって影響を受けます。
溶解性が悪い、Fe の高反応性を考慮した植物組織や器官の間での転送が適切なキレート分子と関連付ける必要があります。また、鉄と鉄のフォーム1の間の酸化還元状態を制御する必要があります。葉、内鉄の約 80% は、電子伝達系、チトクローム、葉緑素と他のヘム分子の生合成に重要な役割はそのための光合成細胞で発見され、鉄 S の形成におけるクラスター7。セル内の余分な鉄の場合、黒字はフェリチン分子8金属が保存されている液胞に転流します。
鉄は、いくつかの方法、含む火炎原子吸収分光法9 (FAAS) または比色試金10, 前者は後者よりもはるかに正確な植物組織で測定できます。FAAS は、個々 の要素の電磁放射に基づくサンプルの元素組成を決定することができる精度の高い手法です。FAAS に変換します金属イオン原子状態のサンプルでは、炎加熱による特定イオンの基底状態に戻るときに特定の波長のイオン励起と放射に 。別のイオンからの排出量は、分光器で区切られ、吸収センサー11によって検出されました。したがって、FAAS は直接鉄濃度を定量化する提供しています。しかし、生体組織中の鉄を可視化するための他の手法が利用できます。誘導結合プラズマ質量分析法 (ICP-MS)12は、鉄などの微量要素を測定するための非常に正確な技術、機器、FAAS と ICP-MS の両方の不足が共通問題。その一方で、チオシアン酸測色13による鉄の測定は、精度を欠いている、サンプル間の小さな変化を検出する失敗します。プルシアン ブルー染色14,15,16,17は鉄フェロシアン化カリウム (K4Fe(CN)6) 生産、鉄イオンとの反応に基づく間接法、青い色の強い、動物や植物の組織の組織学的セクションで質的な鉄の検出に使用されます。
金属 (ゼロ原子価) 鉄は、リソスフェアのまれです。環境中の鉄の支配的な非複合イオンの形態は、ほとんど無酸素環境で比較的より豊富である鉄と鉄有酸素サイトの色合いが強い、周囲の酸素の量によって決まります。鉄酸化の原因となるエージェントはしばしば無酸素、酸性環境18で異なるが、この後者の形式、非常に酸性環境で支配的なも。4% で鉄を溶解するとき HCl (pH 0) 好気性環境で、希薄化後の鉄の主要な部分として存在する、鉄 (Fe3 +)19,20を形成します。
鉄イオンと K4Fe(CN)6の反応は次のとおりです。
Fe3 +: した FeCl3 + K4Fe(CN)6 KFe(III)Fe(II)(CN)6̄ + 3KCl =
Fe2 +: 2 K4Fe(CN)6 + 4 のした FeCl2 = 鉄4(Fe(CN)6)2 + 8 KCl
本研究で我々 はプルシアン ブルー染色することができますソリューションの鉄のレベルを測定するために役立つかどうか尋ねた。
当初は、水溶液中の Fe の濃度とプルシアン ブルー染色の相関関係を検証しました。水溶液中で (した FeCl2、した FeCl3または 2 つの 1:1 混合物) として Fe 濃度は、プルシアン ブルーの付加の後で原子分光法と吸光度 (OD) の両方を測定しました。図 1は、それぞれの方法によって得られる測定の線形回帰曲線を示しています。溶液中の鉄濃度の定量分析のためプルシアン ブルーのメソッドを使用できますと結論づけた。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg"> 図 1: FAAS と光の吸光度によって測定される Fe 濃度の線形回帰 (OD、715 nm) プルシアン ブルーによる得られた。青い正方形とラインは Fe2 +ソリューションを表す、赤い四角形と線 Fe3 +ソリューションおよび黒い正方形を表しライン Fe2 +と Fe3 +の間の 1:1 混合物。次の回帰が得られた: [Fe2 +] = 3 + 123 x 外径 r 0.996、R2 = = 0.989;[Fe3 +] = 1 + 292 x 外径 r = 0.999、R2 = 0.997;[Fe2 +/3 +] = 11 + 146 x 外径 r = 0.983、R2 = 0.956。Fe2 +ドナーした FeCl2 Fe3 +ドナーした FeCl3であった。<a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
比色プルシアン ブルー植物組織の鉄の定量分析法を適応し、火炎吸収原子分光学とプルシアン ブルー染色によりタバコ葉灰の鉄含有量を測定しました。2 つの手法から結果の相関があった。

<table>
	<tbody>
		<tr>
			<td>Potassium Hexacyanoferrate(II)</td>
			<td>Fisher Chemical</td>
			<td>14459-95-1</td>
			<td>Reagent for the Pussian Blue</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter Unit, Vial Vent 0.22 &#956;m</td>
			<td>Millec</td>
			<td>SLGP033RS</td>
			<td>Filter used to filter the ashes + 4% HCl Solution</td>
		</tr>
		<tr>
			<td>Scintillation Vials</td>
			<td>Fisherbrand</td>
			<td>03-337-4</td>
			<td>Used to keep the dry powdered plant material during the burning procedure.</td>
		</tr>
		<tr>
			<td>Disposable Syringe 10 ml</td>
			<td>Medi-Plus</td>
			<td>1931</td>
			<td>Syringe used during the filtration</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>231-595-7</td>
			<td>Used in the 4% HCl solution to dilute the ashes and clean the materials</td>
		</tr>
		<tr>
			<td>Tobacco, Nicotiana tabacum cv. Samsun NN</td>
			<td>Obtained from Prof. Simon Barak and routinely used in the Zaccai Lab</td>
			<td>Barak S, Nejidat A, Heimer Y, Volokita M. Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings. Plant Molecular Biology. 2001 Mar 1;45(4):399-407.</td>
			<td>Tobacco cultivar used in this protocol</td>
		</tr>
		<tr>
			<td>Glass Wool (Rock Wool)</td>
			<td>Sigma-Aldrich</td>
			<td>659997-17-3</td>
			<td>Used in the procedure of burning samples in the furnace.</td>
		</tr>
	</tbody>
</table>

a colorimetric method for measuring iron content in plants

鉄生体内で最も重要な栄養素の一つは、呼吸や光合成などの基本的なプロセスに関与しています。鉄の含有量は乾燥重量の約 0.009% に相当する植物で、すべての生物で幾分低いです。日付、火炎吸収原子分光学植物組織中の鉄濃度を測定する最も正確な方法の一つです。ただし、この方法は時間がかかり、高価な一般的植物研究所は、特定の機器が必要です。したがって、日常的に使用することができます簡単に、まだ正確な方法が必要です。比色のプルシアン ブルー メソッドは、動物や植物の組織切片の染色質の鉄の定期的に使用されます。本研究では、タバコ中の鉄の定量計測法の葉プルシアン ブルーを適応.原子分光学と同じ試料中の鉄の含有量を測定し、線形回帰を発見する染色プルシアン ブルーの両方を使用してこのメソッドの精度を検証しました (R2 = 0.988) 2 つのプロシージャの間。我々 はプルシアン ブルーの植物組織定量的鉄測定法は正確な単純な安価な結論します。しかし、ここで紹介する線形回帰はサンプルと試薬の潜在的な相互作用のための他の植物種のために適切かもしれない。回帰曲線の確立は別の植物種にしたがって必要です。

このプロトコルは正しく実施される、1 つはプルシアン ブルーと原子分光法によって得られた結果と良好な相関性を得る必要があります。したがって、次の実験に反映は、植物試料中の鉄濃度の正確な測定を取得するプルシアン ブルーのメソッドを簡単に使用できます。
タバコがプロトコルに記載を栽培され、異なる鉄?...

Watch this Scientific Journal Video about A Colorimetric Method for Measuring Iron Content in Plants at JoVE.com

A Colorimetric Method for Measuring Iron Content in Plants

植物の鉄含有量を測定するための比色定量法

比色のプルシアン ブルーを用いた植物組織の鉄含有量を測定するための簡潔かつ信頼できるプロトコルを提案します。

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Just medically it can help answer a key question in biology field. Such as how can we easily quantify iron in biology core samples. The main venture of this technique is that it's a easy to produce, and a very precise colorimetric technique.While preferring this technique, you should avoid iron contamination. Visual demonstration of this method will help you to understand its different steps. To begin this procedure, prepare five centimeter by five centimeter pots by filling them with standard pot medium.Plant one tobacco seed in each pot. Transfer the plants to a growth room that is at a constant temperature, under long day conditions of 23 degrees Celsius. Irrigate with tap water until water drains from the pot, and grow the plants for approximately 50 days.After this time, start iron treatments in the irrigation at concentrations appropriate for the experiment. Irrigate the plants with this solution every two days for six to eight days. Then, detach the leaves from the stem by hand, making sure not to use any metal equipment.Using a spray bottle filled with double distilled water, clean each leaf. Dry the leaves on a paper towel, and then transfer them into a paper bag. Place the leaf filled bags in an oven, set to a constant temperature of 80 degrees Celsius, for two to three days.Clean the mortar and pestle twice, with a 4%hydrochloric acid solution. And use a filter paper to dry. Using the mortar and pestle, crush the dry leaves to a powder.Then, transfer the powder to sterile 15 milliliter plastic tubes. First, weigh a new, sealed 20 milliliter scintillation vial without its lid. Either note the weight or tare the scale.Then add the crushed leaves. Weigh the sample in the vial, and note the weight. Use rock wool to close the vial.Weigh three additional vials without adding samples, for use as controls, making note of each weight. Next, transfer the sample and control vials to a furnace. Burn the leaves as outlined in the text protocol.After this, let the samples cool to approximately 100 degrees Celsius. Using heavy gloves and tweezers, remove the samples from the furnace, making sure to hold the vial exteriorly. Place the vials on a flat surface.Remove the rock wool, and close the vials with their original lids. Then, weigh the three control vials, and calculate their average weight gain. If the weight gain is equal to or above 1%of the ash weight, use this value as an estimate of the measurement error.Weigh a 15 milliliter plastic tube. Either record the weight or tare the scale. Then transfer the ashes to the tube.Record this value, which is the ash weight. Next, add five milliliters of the one molar hydrochloric acid solution to the ashes. Filter the ashes through a 22 micrometer filter.Then add an additional five milliliters of the one molar hydrochloric acid solution through the same filter, leading to a final sample volume of 10 milliliters. After this, remove four milliliters from each sample for measurement by atomic spectroscopy. And determine the iron concentration per gram of ash as outlined in the text protocol.To begin, add four grams of potassium ferrocyanide to 100 milliliters of double distilled water, to prepare the Prussian blue solution. Vortex to mix, and store at 4 degrees Celsius until ready to use. When ready to use, mix 50 milliliters of Prussian blue solution with 50 milliliters of one molar hydrochloric acid to serve as the blank solution.Then, use a pipette to mix 0.5 milliliters of a previously obtained sample, with 0.5 milliliters of Prussian blue solution. Let this mixture rest for at least one minute, but for less than five minutes. Transfer this mixture to a cuvette, and use a spectrophotometer to measure the optical density at 715 nanometers.Then, determine the optical density per gram of ash, and plot the linear regression between the iron concentrations, as outlined in the text protocol. The representative results from 21 tobacco leaf samples show that the iron concentration in the irrigation water greatly affected the iron content in the leaf. The spectra of all 21 representative samples are then tested with the Prussian blue method.As seen here, the absorbance at 715 nanometers is the optimal wavelength while measuring solutions containing different concentrations of iron two and iron three. A linear regression curve, which is obtained by plotting iron concentration values obtained by atomic spectroscopy, versus the absorbance values, obtained by the Prussian blue method, allows for new samples of the same plant type to be analyzed. Don't forget that working with HCL can be extremely hazardous.And precaution, such as eye protection, should always be taken while performing this procedure. In the linear regression, it's important to verify that the results are in a realistic range. After its development, this technique can help biology researcher explore additional plants for food production or bioremediation.

a-colorimetric-method-for-measuring-iron-content-in-plants

Research

JoVE Journal

Biochemistry

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

セルロース紙ディスクおよび裸眼比色イムノアッセイにおけるそれらのアプリケーションへの抗体の共有結合

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

生化学

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

RNAの測定方法<em&gt; N</em<sup&gt; 6</sup細胞や組織における&gt; -methyladenosine変更

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

安定同位体のトレースを使用した複雑な細胞のコミュニティのソートされた亜集団で代謝を測定する方法

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

多量-PAGE：生体試料中のキャプチャと解決タンパク質複合体のための方法

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

共沈降によるF-アクチンへのタンパク質結合の測定

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

シアノバクテリアにおけるグリコーゲン含有量の測定

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

マンナン ペースを用いた植物細胞壁多糖の構造解析

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

カエノラブディティス ・ エレガンスにおけるハイスループット化合物スクリーニングのための簡単な方法

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

シアノ バクテリア阻害のフィコビリタンパク質の吸光光度定量

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

測定と酸素消費量全体の解釈飛ぶ頭セグメント

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...