我々は、USGSとバーモント州の水資源と資金を提供するための湖研究センターに感謝します。

1。リン抽出<ol><li> 0.25Mの水酸化ナトリウム（NaOH、40.00グラム/モル）+ 0.05 Mエチレンジアミン四酢酸（EDTA、292.24グラム/モル）の1L溶液を調製します。 </li><li> 50 mLコニカルチューブに濡れたまたは乾燥土壌/サンプル2gのに水酸化ナトリウム+ EDTA溶液30 mLを加え。 </li><li>室温で16時間攪拌しながらインキュベートします。 </li><li> 3分間3000 × gで遠心する。 </li></ol> 2。サンプルの抽​​出pHの調整<ol><li> 1.5 mLのマイクロ遠心チューブに0.1 mLずつを転送します。 </li><li> 2.5 M酢酸0.017 mLを、0.4 M酢酸緩衝液（酢酸ナトリウム[CH 3 COONa、82.03グラム/モル]と酢酸の29.5％、pHは5.0 70.5％）の0.144 mLを加え、脱イオン水の0.739 mLを加え。 </li><li>調整された抽出物の最終容量は1mlになり、最終pHは5.0になります。 </li></ol> 3。酵素のストック溶液の調製<ol><li> 0.1 M酢酸ナトリウム（CH 3 COONa、82.03グラム/モル）溶液、pH 5.0 1 Lを準備します。 </li><li>最終濃度がそれぞれ0.5単位/ mLとなるようにステップ3.1で作成した酢酸ナトリウム緩衝液で2 × 10 mLの酸性ホスファターゼのストック溶液を準備します。 酸性ホスファターゼは、 コムギ （; GP、一般的に0.5 U MG - 1固体、EC 3.1.3.2小麦胚芽）からIを入力 ジャガイモから酸性ホスファターゼタイプIV - S（ジャガイモ、PP、一般的に4.6 U MG - 1固体、EC 3.1.3.2） 活動の*単位は、1ユニットは37℃で時間あたりのソリューションにオルトリン酸の1マイクロモルの解放と定義されているサプライヤー℃で規定されています</li><li>いずれかの溶液に脱イオン水、これが現在4℃で保存できますC今後の使用のため、再構成するには、 ペニシリウムシト （NP1、一般的には500 Uミリグラム-1固体菌類）からヌクレアーゼP1（EC 3.1.30.1）を凍結乾燥する。 </li><li>最終濃度が2.5単位/ mL NP1となるように、ステップ3.2で作成した10 mLのチューブの1本ピペットNP1は、ゆるやかに転倒数回で混ぜる。 </li><li> 2つの10 mLの酵素液を30分間3000 × gで遠心する。 </li></ol> 4。 Pキャリブレーションカーブとコントロール<ol><li> 1 mMのリン酸カリウム（K2HPO4、174.18グラム/モル）ストック溶液の1リットルを準備します。 </li><li> 1.5 mLの遠心管に1 mMリン酸カリウム原液1 mLを追加し、7つの0.5 mLの希釈を行う。 </li><li>を20 nmolの- 0.625 nmolのPからまで7​​段階希釈を持っているように、最初にチューブを捨てる</li><li>標準の96ウェルプレートのH - 行Bに重複して各チューブから80μLを転送する。 </li><li>列の11と12この値が0 nmolのPのキャリブレーションポイントです。の行に酢酸ナトリウム緩衝液（セクション3.1）80μLを追加</li><li>各列11と12は0にナノモルから20 nmolの無機リン酸〜8参照のサンプルが含まれています。 </li><li>列10は、次のコントロールが含まれます。 <ol><li> 10列の最初の3つのウェルに40μlのPP + GPの酵素溶液+ 40μL酢酸ナトリウム緩衝液（セクション3.1）を追加します。 </li><li> 40μLPP + GP + NP1酵素液+次の3つのウエルに40μLの酢酸ナトリウム緩衝液を追加します。 </li><li>井戸一つにし、最終的に10 nmolのグルコース-6リン酸（C 6 H 11のNa 2 O 9 P &amp;bullet; XH 2 O、304.1グラム/モル）+ 40μLPP + GPの酵素溶液を含む40μLの酢酸ナトリウム溶液を加えるコラム10に酢酸ナトリウム溶液の代わりに酵素溶液を加える。 </li></ol></li></ol> 5。サンプル+酵素のインキュベーション<ol><li>テスト中の各サンプルは、標準の96ウェルプレートの1列の最初の9井戸を占有します。 </li><li>最大8行までに第2節から井戸1-9 pH調整サンプルの抽​​出物40μLを配布する。 </li><li>後にすべてのサンプルは*が列4-6および酢酸ナトリウム緩衝液（セクション3.1で調製したもの）にカラム7-9 PP + GP + NP1 ,1 - 3列にPP + GPの酵素溶液を配布するためにマルチチャンネルピペットを使用して配布されている。 *この手順は、すべてのサンプルと同等のインキュベーション時間を取得を保証するために迅速に実行する必要があります。 </li><li>蓋とインキュベートサンプル付き96ウェルプレート+酵素液、コントロール及び検量線37を正確に1時間℃をカバー</li></ol> 6。リリースおよび背景無機Pの比色測定<ol><li>脱イオン水に以下のソリューションの各50 mLを準備します。 ソリューション*：0.1 Mのアスコルビン酸（C6H8O6、176.12グラム/モル）+ 0.5Mトリクロロ酢酸（Cl3CCOOH、163.39グラム/モル） 解決策B：0.01 Mモリブデン酸アンモニウム（（NH4）2MoO4、196.01グラム/モル） 解決策C：0.1 Mクエン酸ナトリウム（HOC（COONa）（CH2COONa）2 2H2O、294.10グラム/モル）+ 0.2 Mのヒ酸ナトリウム（NaAsO2、129.91グラム/モル）+ 5％氷酢酸 （CH3CO2H、60.05グラム/モル）*ソリューションは、毎日準備しておく必要があります。 </li><li> 96ウェルPLで全てのウェルにB液、50μLのと溶液Cの、20μLソリューションSDS、液100μLの25μLを追加食べた。マルチチャンネルピペットで急速にこれを行います。 </li><li>プレートを覆い、室温で30分間インキュベートする。 </li><li>任意のチューナブルマイクロプレートリーダーで850 nmで吸光度を測定します。 </li></ol> 7。 P化合物の分類<ol><li>スプレッドシートアプリケーション（Microsoft Excelなど）に生データをインポートします。 </li><li> x軸に0から40 nmolのPとy軸上の重複した吸光度測定値の平均：無機リンの検量線をプロットします。 </li><li>線形回帰を実行し、最適の線の方程式を見つける。 </li><li> 1から3列：直接検量線 - これはバックグラウンド無機Pの適用</li><li>列4-6：平均三連のサンプル値とP加水分解性Pのモノエステルから誘導されるグロス - これは吸光度からコントロール（酵素溶液+背景の無機P）を差し引く。 </li><li>列7-9：列4-6減算の吸光度値を除いて7.5と同じです - これは加水分解性PのジエステルからPです。 </li><li>校正式を適用してリリースさnmolのPへの吸光度データを変換します。 </li><li> nmolのPはmg P /元土壌のkgに戻って関連することができますリリース。また、各Pのクラスの割合を評価することも、データを分析するための有用なアプローチかもしれません。 </li></ol> 8。代表的な結果： 比色化学後に96ウェルプレートの迅速な目視検査は、手順が正しく行われたか否かに手がかりを提供します。最初に確認することは板の側面のプロフィールをスキャンすることによって、各ウェル中の液体のレベルです。すべてのウェルに試薬を正確に275μLがあるはずです。次に、視覚的に三連のサンプルウェルと重複してキャリブレーションの井戸の色を検査する。これらの技術的複製は、青の同じ色でなければなりません。次のグルコース6リン酸を含む二つのウェルに検量線を適用し、それらが全体の抽出されたPは、ICP - OESまたは別の方法を用いて測定された後、これを用いて計算された合計Pが値を確認して無機Pの全10 nmolのリリースを確認してくださいプロトコルは、抽出されたPの量を超えないようにしてください。 注：スプレッドシート内の慎重なデータ管理は定量的なエラーを防ぐ助けになる。一度に数が多いに対処されるので、テンプレートを作成することは十分に費やした時間となります。 <img src="/files/ftp_upload/2660/2660fig1.jpg" alt="図1" /> 図1 8つのサンプル（行AH）と較正曲線（カラム11および12）の結果を示す96ウェルプレート。コントロールは列10になります。列1から3および4-6間の色の強度の増加は、列4-6と7月9日の間に、加水分解モノエステルのP化合物が原因である加水分解ジエステルのP化合物によるものです。 <img src="/files/ftp_upload/2660/2660fig2.jpg" alt="図2" /> 図2ハイスループット酵素加水分解を使用してバーモント州の土壌試料の0.25 M水酸化ナトリウム- 0.05MのEDTA抽出物中のPクラスの分布。エラーバーは標準偏差、n = 3のを示している。

<ol>
	<li>Turner, B. L., Frossard, E., Baldwin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organic phosphorus in the environment. , (2005).</li><li>Murphy, J., Riley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+single+solution+method+for+determination+of+phosphate+in+natural+waters.">A modified single solution method for determination of phosphate in natural waters.</a> Anal. Chim. Acta. 27, 31-36 (1962).</li><li>Dick, W. A., Tabatabai, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+orthophosphate+in+aqueous+solutions+containing+labile+organic+and+inorganic+phosphorus+compounds.">Determination of orthophosphate in aqueous solutions containing labile organic and inorganic phosphorus compounds.</a> J. Environ. Qual. 6, 82-85 (1977).</li><li>He, Z., Toor, G. S., Honeycutt, C. W., Sims, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+hydrolysis+approach+for+characterizing+labile+phosphorus+forms+in+dairy+manure+under+mild+assay+conditions.">An enzymatic hydrolysis approach for characterizing labile phosphorus forms in dairy manure under mild assay conditions.</a> Bioresour. Technol. 97, 1660-1668 (2006).</li><li>He, Z., Honeycutt, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+molybdenum+blue+method+for+orthophosphate+determination+suitable+for+investigating+enzymatic+hydrolysis+of+organic+phosphates.">A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates.</a> Commun. Soil Sci. Plant Anal. 36, 1373-1383 (2005).</li><li>Cade-Menun, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+phosphorus+in+environmental+and+agricultural+samples+by+31P+nuclear+magnetic+resonance+spectroscopy.">Characterizing phosphorus in environmental and agricultural samples by 31P nuclear magnetic resonance spectroscopy.</a> Talanta. 66, 359-371 (2005).</li><li>Johnson, N. R., Hill, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorus+species+composition+of+poultry+manure-amended+soil+using+high-throughput+enzymatic+hydrolysis.">Phosphorus species composition of poultry manure-amended soil using high-throughput enzymatic hydrolysis.</a> Soil Sci. Soc. Am. J. 74, 1786-1791 (2010).</li></ol>

利害の衝突は宣言されません。

まさにその性質によって、少量を使用して迅速な方法は、細心の注意が必要です。したがって、最も重要なステップは、プレート上にピペットのソリューションを含むものです。正確な、そして最も重要なことは、一貫性のあるピペットのテクニックは、このアッセイの成功に不可欠です。 水酸化ナトリウム- EDTAの抽出は、多くの試料のPの大部分は三つのクラスに特徴付けできるようになります：オルトリン酸、モノエステルPおよびジエステルP.土壌、肥料、堆積物又はNaOH - EDTA -抽出Pを含む任意の他の環境サンプルを特徴づけることができる。環境試料中のP形は必ずしも安定ではなく、この手法は、サンプルは研究者の軍隊を採用することなく、危険にさらされる前にサンプルが特徴とされていることを確認します。 多数のサンプルが処理されるときに、このアッセイは特に適しています。試薬の必要性と容量の要件は、管理可能なレベル（例えば、1.5 mlのマイクロチューブではなく、50mLのガラス製のフラスコ）にスケールダウンされています。この適応はまた、廃棄物の生産を制限する。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> NaOHで</td><td>シグマアルドリッチ</td><td> S8045 </td></tr><tr><td> EDTA </td><td>シグマアルドリッチ</td><td> EDS </td></tr><tr><td>氷酢酸</td><td>シグマアルドリッチ</td><td> 242853 </td></tr><tr><td>酢酸ナトリウム</td><td>シグマアルドリッチ</td><td> S2889 </td></tr><tr><td>小麦の酸性ホスファターゼ</td><td>シグマアルドリッチ</td><td> P3627 </td></tr><tr><td>ポテト酸性フォスファターゼ</td><td>シグマアルドリッチ</td><td> P1146 </td></tr><tr><td>ヌクレアーゼP1 </td><td>シグマアルドリッチ</td><td> N8630 </td></tr><tr><td>リン酸カリウム</td><td>シグマアルドリッチ</td><td> P2222 </td></tr><tr><td></td><td>シグマアルドリッチ</td><td></td></tr><tr><td></td><td>シグマアルドリッチ</td><td></td></tr><tr><td>グルコース-6リン酸</td><td>シグマアルドリッチ</td><td> G7250 </td></tr><tr><td> SDS </td><td>シグマアルドリッチ</td><td> L4390 </td></tr><tr><td>アスコルビン酸</td><td>シグマアルドリッチ</td><td> A5960 </td></tr><tr><td> TCA </td><td>シグマアルドリッチ</td><td> T9159 </td></tr><tr><td>モリブデン酸アンモニウム</td><td>シグマアルドリッチ</td><td> A1343 </td></tr><tr><td>クエン酸ナトリウム</td><td>シグマアルドリッチ</td><td> S1804 </td></tr><tr><td>ヒ酸ナトリウム</td><td>シグマアルドリッチ</td><td> S9663 </td></tr></tbody></table>

high-throughput measurement and classification of organic p in environmental samples

有機燐（P）は、分子の多くの種類は、環境試料1に存在します。彼らは比色試薬2,3と反応しないため、従来のPの測定は、これらの有機P化合物を検出しません。酵素加水分解は、（EH）正確に4,5の環境試料中の有機Pの形態を特徴づけるための新しい方法です。このメソッドは、リン- 31核磁気共鳴分光法（31 P - NMR）、高価であり、専門的な技術training6を必要とするメソッドによって精度に捏造されています。我々は、マイクロプレートリーダーシステム7にリンの3つのクラス（モノエステルP、ジエステルP及び無機P）を測定できる酵素加水分解法を適応している。このメソッドは、土壌、堆積物、肥料や、場合集中、水生サンプルでPの種を測定するために、高速で正確、​​手頃な価格とユーザーフレンドリーな手段を研究者に提供します。これは標準の実験室で行うことができる有機Pの形態と酵素不安定性を測定するための唯一のハイスループット法である。結果のデータは、システム栄養成分と富栄養化の可能性を研究する科学者に洞察力を提供します。

Watch this Scientific Journal Video about High-Throughput Measurement and Classification of Organic P in Environmental Samples at JoVE.com

High-Throughput Measurement and Classification of Organic P in Environmental Samples

このプロトコルは、96サンプルのPモノエステル、Pのジエステルと無機P.アップが標準的な実験で一度に測定できるように土壌のリンを測定し、分類するためのマイクロプレートリーダーを利用して酵素加水分解のハイスループット法を説明します。

環境試料中の有機Pのハイスループット測定と分類

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

high-throughput-measurement-classification-organic-p-environmental

Research

JoVE Journal

Biology

12.5K ビュー.  University of Vermont. このプロトコルは、96サンプルのPモノエステル、Pのジエステルと無機P.アップが標準的な実験で一度に測定できるように土壌のリンを測定し、分類するためのマイクロプレートリーダーを利用して酵素加水分解のハイスループット法を説明します。

ビデオ: High-Throughput Measurement and Classification of Organic P in Environmental Samples

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

における真菌エンドグルカナーゼ活性のハイスループットスクリーニング大腸菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

粉末試料の絶対量子収率測定

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

複雑なサンプル中の特定のタンパク質のグリコシル化の多重化ハイスループットプロファイリングのための化学的にブロックされた抗体マイクロアレイ

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

細菌の増殖の高精度、高スループットの分析

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

フローサイトメトリーによる細胞性粘菌キイロタマホコリカビマクロピノサイトーシスの高スループット測定

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

ゼブラフィッシュ稚魚を用いた消化管通過時間の高スループット測定

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;