私たちは、シアノバクテリア菌株を提供するために大学自治・デ・マドリードから博士アントニオ・ケサダに感謝します。この作品は、スペイン語MINISTERIOデエコノミアのy Competitividad（MINECO）のSubdirección一般デProyectosデInvestigaciónによって資金を供給された、無許可します。 AYA2011-24803とESP2014-58494-R。

<ol>
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The authors have nothing to disclose.

ここでは、CYANOCHIP、シアノバクテリア属の広い範囲の検出および同定のための17-抗体マイクロアレイを用いて、多重蛍光サンドイッチ免疫測定法は、22に記載されています。これらのシアノバクテリアは、そのうちのいくつかは毒素生産され、淡水生息地の中で最も頻繁に底生やプランクトンの属を表します。最近、蛍光サンドイッチイムノアッセ?...

複雑な自然の微生物群集中の微生物の検出と監視は生物医学、環境、エコロジー、そして宇宙生物学などの多くの分野で重要です。淡水中の細胞の花（過剰増殖）を形成する能力についてよく知られている原核微生物はシアノバクテリアです。彼らは遍在している、と多くの種は、ヒトの健康に対する潜在的なリスクに、だけでなく、生態系への影響のみならず大手、毒素を産生することができます。この点では、 シアノバクテリアおよび/または土壌及び水中のそれらの毒素の早期検出のための迅速かつ高感度な方法を開発することが不可欠です。水管理者が適切な水管理プログラムを実施する際に、結果的に、決定にそれらを助けるために、この目的のために、多重蛍光サンドイッチマイクロアレイイムノアッセイ（FSMI）のツールとして開発されました。 方法の多様な範囲は、シアンを検出および同定するために開発されています光学顕微鏡、分子生物学、および免疫学的技術を含む土壌や水にobacterial細胞およびシアノトキシン、。これらの方法は、それらが提供する情報に大きく異なります。顕微鏡技術は、細胞の形態や、フィコシアニンやクロロフィル1とシアノバクテリア顔料、からの生体内蛍光の検出に基づいています。彼らは、試料中に存在するシアノバクテリアの種類と数をお知らせリアルタイムおよび頻繁なモニタリングのための迅速かつ安価な方法であるが、それらは潜在的な毒性に関する情報を与えることはありません。さらに、彼らは、しばしば密接に関連した種2を区別することは非常に困難であることを考慮して、専門知識の特定のレベルを必要とします。これらの制限を克服するために、光学顕微鏡は、生物学的および生化学的スクリーニングアッセイおよびシアノトキシンの同定および定量のための物理化学的方法の両方を添付しなければなりません。 <pクラス= &quot;jove_content&quot;&gt;酵素結合免疫吸着検定法（ELISA）、タンパク質リン阻害アッセイ（PPIA）、およびマウスにおける神経化学的試験は、シアノトキシンを検出するための生化学的スクリーニングアッセイの例です。最初の二つは、迅速かつ高感度な方法論であるが、使用してELISAおよびPPIAテストは毒素の3種類に制限されている場合、偽陽性が記載されています。マウスバイオアッセイは、低感度および精度の質的な技術であり、特別なライセンスやトレーニングが必要です。また、試料中に存在する毒素の種類についての情報を与えません。シアノトキシン例えば、高速液体クロマトグラフィー（HPLC）などの他の分析方法によって同定及び定量することができる、液体クロマトグラフィー - 質量分析（LC-MS）、ガスクロマトグラフィー（GC）、ガスクロマトグラフィー - 質量分析（GC-MS）またはマトリックス支援レーザー脱離/イオン化飛行時間（MALDI-TOF）。参照標準、必要とされる場合は、これが唯一の可能性であります複雑なサンプル中の個々の毒素濃度を決定するためのedは、3、4ご利用いただけます。また、これらの方法は時間がかかります。高価な機器、消耗品、およびサンプル調製を必要とします。そして、経験豊富な専門スタッフによって行われなければなりません。 分子ベースの方法は、ゲノムデータベースに公開された配列情報のおかげで、検出、同定、およびシアノバクテリアとそれらに対応するシアノトキシンを定量化するために数十年にわたって適用されている（ 例えば、バイオテクノロジー情報、NCBIのための国立センター）。これらの方法の中でDNAの増幅のためのプライマーセットの設計を必要とし、別のシアノバクテリア種のDNA配列の予備知識に依存してポリメラーゼ連鎖反応（PCR）に基づくものです。遺伝子の検出は、フィコシアニンオペロンのように、属レベルでの正確な同定をもたらす一方で、一部の種や菌株がで検出されませんこの方法。しかしながら、このようなミクロオペロンに属するものとして毒素をコードする遺伝子は、プロデューサ5不足しているサンプル中の毒素の同定を容易にします。それにもかかわらず、PCRによる毒素マーカーの検出は、必ずしも環境に毒性を示すものではありません。また、 シアノバクテリアの種と試料中に存在する毒素生産の全範囲を分析するために開発されたプライマーのセットは、まだ不完全であり、更なる研究は、未知の種を同定するために行われなければなりません。他の分子技術は、 蛍光in situハイブリダイゼーション（FISH）およびDNAマイクロアレイなどの非PCRベースです。 過去20年間では、マイクロアレイ技術は、特に環境モニタリングでは、アプリケーションの多くの分野で重要性を増しています。 DNAマイクロアレイは、種との検体4の間の識別、6、7を可能に</SUP&gt;、8、9、10、それらは非常に面倒で時間がかかる複数のステップ（ 例えば、マイクロアレイの性能、DNA抽出、PCR増幅、およびハイブリダイゼーション）を伴う作業を考えています。そのため、例えば、サンドイッチおよび競合免疫学的マイクロアレイのような抗体に基づいてより少ない時間のかかるアッセイは、多数の環境の分析物11、12、13の検出のために不可欠で信頼性の高いハイスループット法となっています。抗体の能力は、特に、それらの標的化合物を認識し、ほとんどすべての物質に対する抗体を産生する可能性とともに、検体およびタンパク質の少量を検出するために、抗体マイクロアレイを環境目的のための強力な技術を作ります。また、複数を達成する能力は、として分析しますイングルアッセイは、ppmのppbの範囲の検出限界で、この方法14の主な利点の1つです。 抗体ベースのバイオセンサーは、環境モニタリング15、16、17、18、19、20、21中の病原体および毒素の広い範囲を検出するための高感度かつ迅速なツールであることが判明しています。 DNA法は、いくつかのステップを含むが、抗体ベースのマイクロアレイは、主に適切なソリューションバッファ内の短い溶解段階に基づいて、小さなサンプル調製を必要とします。 DelehantyとLigler 15 4 PPBのANのタンパク質濃度を検出することができる抗体サンドイッチ免疫測定法に基づいて、複雑な混合物中のタンパク質および細菌分析物の同時検出を報告しD 10細胞の4 CFU / mLです。 Szkola ら。 21は生物兵器に使用されるかもしれないproteotoxinsと小さな毒素、化合物の同時検出のための安価で信頼性の高いマルチプレックスマイクロアレイを開発しました。彼らは20分未満で、3 ppbでの検出限界で、リシン毒素の濃度を検出しました。最近、CYANOCHIP、毒性および非毒性のシアノバクテリアのin situ検出のための抗体マイクロアレイベースのバイオセンサーは、22に記載されています。このマイクロアレイは、主に顕微鏡的に識別することが困難である水生環境で、潜在的なシアノバクテリアブルームの同定を可能にします。でも、種レベルで、多重検出およびシアノバクテリアの同定のための費用対効果の高いツールにこのバイオセンサーを回すほとんどの種のために10 3細胞、 -マイクロアレイの検出限界は10 2です。これらのプロパティはすべてantiboを作りますDYマイクロアレイ技術、およびこの仕事で提示特に方法、前述の技術に比べて迅速かつ簡単な方法。 この作品は、土壌と水のサンプル中のシアノバクテリアの存在を検出するために抗体マイクロアレイベースのバイオセンサーを使用した実験の2つの例を示します。これは、非常に少量の試料と非常に基本的なサンプル調製を必要とするサンドイッチイムノアッセイフォーマットに基づいて簡単かつ信頼できる方法です。この方法は、短い時間を必要とし、容易フィールドで行うことができます。

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">0.22 mm pore diameter filters</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">GSWP04700</td>
			<td style="width:427px;">For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Eppendorf&#160; 5424R microcentrifuge</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Phosphate buffer saline (PBS) pH 7.4 (10X)</td>
			<td style="width:248px;">Thermofisher Scientific</td>
			<td style="width:119px;">70011036</td>
			<td>50 mM potassium phosphate, 150 mM NaCl, pH 7.4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Ultrasonic processor UP50H</td>
			<td style="width:248px;">Hielscher</td>
			<td style="width:119px;"></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Complete Freund&#39;s adjuvant&#160;</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">F5881</td>
			<td>Immunopotentiator</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Incomplete Freud&#39;s adjuvant</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">F5506</td>
			<td>For boost injections</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protein A antibody purification kit&#160;&#160;</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">PURE1A&#160;</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Centrifugal filter devices MWCO&#60;100 KDa</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">UFC510096-96K</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Dialysis tubings, benzoylated</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">D7884-10FT</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Illustra Microspin G-50 columns&#160;</td>
			<td style="width:248px;">GE-HealthCare</td>
			<td style="width:119px;">GE27-5330-02</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Bradford reagent</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">B6916-500 mL</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">MicroBCA protein assay kit&#160;</td>
			<td style="width:248px;">Thermo Scientific</td>
			<td style="width:119px;">23235</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protein arraying buffer 2X</td>
			<td style="width:248px;">Whatman (Sigma Aldrich)&#160;</td>
			<td style="width:119px;">S00537</td>
			<td>Printing buffer; 30-40% glycerol in 1X PBS with 0.01% Tween 20</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Tween 20&#160;</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">P9416</td>
			<td>Non-ionic detergent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Bovine serum albumin (BSA)</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">A9418</td>
			<td>Control&#160; for printing; blocking reagent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">384-wells microplate</td>
			<td style="width:248px;">Genetix</td>
			<td style="width:119px;">X6004</td>
			<td>For antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Robot arrayer for multiple slides</td>
			<td style="width:248px;">MicroGrid II TAS arrayer from Digilab</td>
			<td style="width:119px;"></td>
			<td>For antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Epoxy substrate glass slides</td>
			<td style="width:248px;">Arrayit corporation</td>
			<td style="width:119px;">VEPO25C</td>
			<td>Solid support for antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Alexa Fluor-647 Succinimidyl-ester&#160;</td>
			<td style="width:248px;">Molecular probes</td>
			<td style="width:119px;">A20006</td>
			<td>Fluorochrome</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">DMSO&#160;</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">D8418</td>
			<td>Fluorochrome dissolvent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heidolph Titramax vibrating platform shaker</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For antibody labeling&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Illustra Microspin G-50 columns&#160;</td>
			<td style="width:248px;">Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
			<td>For purification of labeled antibodies</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Safe seal brown 0,5 ml tubes</td>
			<td style="width:248px;">Sarstedt</td>
			<td style="width:119px;">72,704,001</td>
			<td>For labeled antibodies storage&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Nanodrop 1000 spectrophotometer</td>
			<td style="width:248px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>To quantify antibody concentration and labeling efficiency</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">3 &#181;m pore size polycarbonate 47 mm diameter filter</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">TMTP04700</td>
			<td>To concentrate cells</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">1M Trizma hydrochloride solution pH 8</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T3038</td>
			<td>For TBSTRR preparation; to block slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium chloride</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">S7653</td>
			<td>For TBSTRR preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">20 &#181;m nylon filters&#160;</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">NY2004700</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">10-12 mm filter holders</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">SX0001300</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protease inhibitor cocktail</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">P8340</td>
			<td>For environmental extract storage</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">1M Trizma hydrochloride solution pH 9</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T2819</td>
			<td>To block slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heidolph Duomax 1030 rocking platform shaker</td>
			<td style="width:248px;">VWR</td>
			<td style="width:119px;"></td>
			<td>To block slides; for incubation processes&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">VWR Galaxy miniarray microcentrifuge</td>
			<td style="width:248px;">VWR</td>
			<td style="width:119px;">C1403-VWR</td>
			<td>To dry slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Multi-Well microarray hybridization cassette</td>
			<td style="width:248px;">Arrayit corporation</td>
			<td>AHC1X24</td>
			<td>Cassette for 24 assays per slide</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">GenePix 4100A microarray scanner&#160;</td>
			<td style="width:248px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Scanner for fluorescence</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">GenePix Pro Software</td>
			<td style="width:248px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Software for image analysis and quantification</td>
		</tr>
	</tbody>
</table>

experimental protocol for detecting cyanobacteria in liquid and solid samples with an antibody microarray chip

地球温暖化や富栄養化は、いくつかの水生生態系が急速かつ大規模なシアノバクテリアの成長をトリガとして、真のバイオリアクターを動作させます。これは、関連する健康と経済的な影響を持っています。多くのシアノバクテリア菌株は毒素生産者である、とだけ少数の細胞は、環境に取り返しのつかない損傷を誘発するために必要です。したがって、水体当局との投与は、それらの予防的もしくは治療的な意思決定をサポートするために信頼できるデータを提供する迅速かつ効率的な早期警報システムを必要とします。この原稿は、分類学上の解像度（CYANOCHIP）で17抗体（ABS）を有する抗体マイクロアレイチップを使用して、毒素産生シアノバクテリア株のインフィールド検出のための実験プロトコルを報告します。ここでは、17シアノバクテリア株の同時モニタリングのための多重蛍光サンドイッチマイクロアレイイムノアッセイ（FSMI）が頻繁に淡水生態系に咲く発見、それらのいくつかの毒素生産者は、記載されています。マルチでのマイクロアレイPLE CYANOCHIPの（24まで）同一の複製を同時にサンプルの同様の数をテストするために、単一の顕微鏡スライド上に印刷しました。液体試料は、1〜3ミクロンのフィルターを通して濾過することにより、抗体との直接的インキュベーション（ABS）または細胞濃度後のいずれかで試験することができます。そのような堆積物とグランド石のような固体サンプルを、最初のインキュベーション緩衝液中のハンドヘルド超音波処理によって均質化し、分散しています。粗物質を除去し、ろ液をABSでインキュベートする - それらは、次いで、（20ミクロン5）濾過します。免疫反応は、17蛍光標識Abの混合物で最終インキュベーションによって明らかにされており、可搬型蛍光検出器によって読み取られます。全体のプロセスは、そのほとんどが、インキュベーションの2 1時間の期間に対応し、約3時間かかります。出力は、明るいスポットは、シアノバクテリアマーカーの陽性検出に対応した画像です。

この作品はCYANOCHIP抗体マイクロアレイを用いて、最も関連性の高い淡水シアノバクテリア種の同時同定（ 表1）のためのマルチプレックスイムノアッセイテストを説明します。マイクロアレイは、顕微鏡スライド上に印刷された3×8マイクロアレイフォーマットすることができます。各マイクロアレイは、陰性対照としてトリプリケートスポットパターン?...

Watch this Scientific Journal Video about Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip at JoVE.com

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

検出するための実験プロトコール<em&gt;シアノバクテリア</em&gt;抗体マイクロアレイチップと液体と固体試料中の

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

experimental-protocol-for-detecting-cyanobacteria-liquid-solid

Research

JoVE Journal

Environment

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

9.0K ビュー.  Centro de Astrobiología (CAB, INTA-CSIC). The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

ビデオ: Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

の操作植物に誘導される土壌の不均一性のための実験プロトコール

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

環境学

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

小さな汚染物質の免疫学的分析のための水試料の自動固相抽出ための簡単​​な方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ecological interactions. Arthropods are usually tracked in nature by tagging them with a unique mark and then re-collecting them over time and space to determine their dispersal capabilities. In addition to actual physical tags, such as colored dust or paint, various types of proteins have proven very effective for marking arthropods for ecological research. Proteins can be administered internally and/or externally. The proteins can then be detected on recaptured arthropods with a protein-specific enzyme-linked immunosorbent assay (ELISA). Here we describe protocols for externally and internally tagging arthropods with protein. Two simple experimental examples are demonstrated: (1) an internal protein mark introduced to an insect by providing a protein-enriched diet and (2) an external protein mark topically applied to an insect using a medical nebulizer. We then relate a step-by-step guide of the sandwich and indirect ELISA methods used to detect protein marks on the insects. In this demonstration, various aspects of the acquisition and detection of protein markers on arthropods for mark-release-recapture, mark-capture, and self-mark-capture types of research are discussed, along with the various ways that the immunomarking procedure has been adapted to suit a wide variety of research objectives.

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

管理と分散研究のための節足動物にプロテインマークを検出

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

コマーシャルからの副産物としてアルケノンの単離とバイオディーゼル生産のための実験プロトコール<em&gt;イソクリシス</em&gt;藻類バイオマス

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

多数の試料を用いた実験における土壌微生物の同定のための脂質抽出と分析法

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Experimental System of Solar Adsorption Refrigeration with Concentrated Collector

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The main components of the system were the adsorbent bed, the condenser, the evaporator, the cooling sub-system, and the solar collector. In the first step of the experiment, the vapor-saturated bed was heated by the solar radiation under closed conditions, which caused the bed temperature and pressure to increase. When the bed pressure became high enough, the bed was switched to connect to the condenser, thus water vapor flowed continually from the bed to the condenser to be liquefied. Next, the bed needed to cool down after the desorption. In the solar-shielded condition, achieved by aluminum foil, the circulating water loop was opened to the bed. With the water continually circulating in the bed, the stored heat in the bed was took out and the bed pressure decreased accordingly. When the bed pressure dropped below the saturation pressure at the evaporation temperature, the valve to the evaporator was opened. A mass of water vapor rushed into the bed and was adsorbed by the zeolite material. With the massive vaporization of the water in the evaporator, the refrigeration effect was generated finally. The experimental result has revealed that both the COP (coefficient of the performance of the system) and the SCP (specific cooling power of the system) of the SAPO-34 zeolite was greater than that of the ZSM-5 zeolite, no matter whether the adsorption time was longer or shorter. The system of the SAPO-34 zeolite generated a maximum COP of 0.169.

With solar energy as the driving force, a novel adsorption refrigeration system has been developed and experimentally investigated. Water vapor and zeolite formed the working pair of the adsorption system. This manuscript describes the setup of the experimental rig, the operation procedure, and the important results.

太陽吸着冷凍濃縮コレクターの実験システム

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

有機塩素系農薬に曝露した肝細胞におけるミトコンドリア機能の検出実験プロトコル

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

ほこりの充電と紫外放射またはプラズマへの露出表面上動員の実験方法

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

最適化された小型リン定量法を用いた粒状水酸化第二鉄に酸の吸着を確認する手順

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

植物感染試験: スプレーと傷を介した植物病原体イネいもち病菌接種

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

検出するための実験プロトコール

要約

さらに動画を探す

この動画の章

の操作植物に誘導される土壌の不均一性のための実験プロトコール

小さな汚染物質の免疫学的分析のための水試料の自動固相抽出ための簡単な方法

管理と分散研究のための節足動物にプロテインマークを検出

コマーシャルからの副産物としてアルケノンの単離とバイオディーゼル生産のための実験プロトコール

多数の試料を用いた実験における土壌微生物の同定のための脂質抽出と分析法

太陽吸着冷凍濃縮コレクターの実験システム

有機塩素系農薬に曝露した肝細胞におけるミトコンドリア機能の検出実験プロトコル

ほこりの充電と紫外放射またはプラズマへの露出表面上動員の実験方法

最適化された小型リン定量法を用いた粒状水酸化第二鉄に酸の吸着を確認する手順

植物感染試験: スプレーと傷を介した植物病原体イネいもち病菌接種

検出するための実験プロトコール

要約

さらに動画を探す

この動画の章

の操作植物に誘導される土壌の不均一性のための実験プロトコール

小さな汚染物質の免疫学的分析のための水試料の自動固相抽出ための簡単​​な方法

管理と分散研究のための節足動物にプロテインマークを検出

コマーシャルからの副産物としてアルケノンの単離とバイオディーゼル生産のための実験プロトコール

多数の試料を用いた実験における土壌微生物の同定のための脂質抽出と分析法

太陽吸着冷凍濃縮コレクターの実験システム

有機塩素系農薬に曝露した肝細胞におけるミトコンドリア機能の検出実験プロトコル

ほこりの充電と紫外放射またはプラズマへの露出表面上動員の実験方法

最適化された小型リン定量法を用いた粒状水酸化第二鉄に酸の吸着を確認する手順

植物感染試験: スプレーと傷を介した植物病原体イネいもち病菌接種

小さな汚染物質の免疫学的分析のための水試料の自動固相抽出ための簡単な方法