Name: a method for extracting pigments from squid doryteuthis pealeii
Uploaded: 2016-11-09T15:00:00.000+00:00
Duration: 11 min 3 s
Description: Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

本明細書中で行わ無脊椎動物動物研究は、米国では規制されていません。したがって、制度的動物実験委員会は、そのようなプロトコルの見直しのための権限を有しません。これらの代わりに、米国における規制の管轄下に落下する著者がここにこれらの研究は、誠実な倫理的な利用に向けた努力、ケア、およびこれらの動物の治療で行われたと述べている、個人の数が最小化され、これらの努力ではありませんバーゼル宣言と実験動物学のための国際評議会（ICLAS）倫理ガイドラインと一致しています。 1. Doryteuthis pealeii（D.のpealeii）解剖<ol><li>購入断頭イカD.ウッズホール、マサチューセッツ州で海洋生物学研究所で海洋資源省から、または任意の新鮮な魚市場からpealeii。フィレットまたは以前にこの手順を適用するために変更された検体は使用しないでください。具体的には、彩度tophore層は依然として、試料上無傷でなければなりません。 <ol><li>動物をすぐに使用しない場合は、使用するまで-20℃で保存します。標本を解凍するには、前の解剖に1時間室温の水にそれらを配置します。 </li></ol></li><li>動物の後側に沿って切断したステンレス鋼ストレート細かい点解剖ハサミを使用。腹マントルの底から始まり、フィン領域に終わります。 </li><li>鉗子を解剖し、解剖ハサミを使用して結合組織を切断して、それらを持ち上げることによって内臓（胃、鰓、インク腺、生殖器官、全身心臓、および上腕心臓）のすべてを削除します。標本袋に臓器を捨てます。 </li><li>柔軟な解剖パッドを含む標準11-1 / 2 &quot;×7-1 / 2&quot;×1-1 / 2 &quot;アルミ解剖パンにマントル（フィン側まで）をピンに解剖T-ピンを使用してください。組織水没使い捨てポリスチレン真空濾過システムを通して濾過された海水を250ml0.2μmの細孔サイズを含みます。 </li><li>慎重に無傷の皮膚層を含む色素胞を保ち、解剖鉗子で表皮層（不透明色）を取り外します。表皮層が除去されると、試料袋に廃棄します。 </li><li>鉗子や解剖ハサミで色素胞層の小さい（1センチメートル×1センチ）のセクションを分析。 1.5ミリリットルマイクロ遠心チューブ内のサンプルを収集する（解剖組織と各空のチューブの半分を埋めます）。 </li><li>チューブを含む組織にフィルタ海水の500μLを加えます。サンプルを、4℃で一晩保存することができます。 </li></ol> 2.単離色素胞色素顆粒<ol><li> 組織消化 <ol><li>パパイン/コラゲナーゼの調製<ol><li>紙の重さ4 &quot;×4&quot;低窒素を使用してコラゲナーゼを30mgおよびパパイン5mgを秤量。スクリューキャップを有する50mLのポリプロピレン遠心管にこれらを移します。 </li><li> deionizの10ミリリットルを測定します目盛り付きピペットを用いて、ED水。パパイン/コラゲナーゼを含むバイアルに直接追加します。最高の設定上の渦溶液が透明になるまで。 </li></ol></li><li>細胞外組織を削除します<ol><li>遠心分離14000×gで5分間のステップ1.7から収集した色素胞組織。廃プラスチックのバケツに海水のトップ層を捨てます。 </li><li> P1000容量可変シングルチャンネルマイクロピペットを用いて組織にパパイン/コラゲナーゼ溶液500μlを追加します。最高設定に1-2分間、各サンプルをボルテックスし。 </li><li>周囲の組織から細胞を解離するためには40kHzの周波数で5分間サンプルを超音波処理します。 </li><li> 14,000×gで5分間遠心分離します。廃プラスチックのバケツに上清を捨てます。 </li><li> G X 14,000で5分間収集色素胞組織を遠心。廃プラスチックのバケツに海水のトップ層を捨てます。もう一度繰り返します。 </li><li>手動で任意の残りの大規模な組織秒を削除次のステップに進む前に、ピンセットで、このプロセスに分解しなかったン。 </li></ol></li></ol></li><li> 顔料顆粒の単離 <ol><li>均質化緩衝液の調製<ol><li> （4-（2-ヒドロキシエチル）-1-ピペラジンエタンスルホン酸）（HEPES、100mMの）、塩化マグネシウム（10ミリモル）0.095 gのカリウム、アスパラギン酸（50ミリモル）の0.856 g及び0.015gの2.38グラム計量ジチオスレイトール（1 mM）の。スクリューキャップと150ミリリットルのポリスチレン容器に移します。 1商業ミニタブレットのプロテアーゼ阻害剤を追加します。 </li><li>メスシリンダーを用いて、100mlの脱イオン水を測定します。均質化塩を含有する容器に直接追加します。解決までの渦は明らかです。 </li></ol></li><li>顔料顆粒の均質化<ol><li> 5分間、14,000×gでステップ2.1から各サンプルを遠心分離します。廃プラスチックのバケツに上清を捨てます。このサンプルでは、​​細胞外組織の空隙でなければなりません。 </li><li> 500＆を追加＃181; 1〜2分間ごとに、各チューブとボルテックスに均質化緩衝液のリットルは徹底的にサンプルを混合します。 30分（〜40 kHzの）のためのサンプルを超音波処理し、14,000×gでの遠心分離の5分に従ってください。 </li><li>廃プラスチックのバケツに上清を捨て、そして色素胞の球形嚢とタンパク質テザーの完全な消化を確実にするために、前のステップを2-3回繰り返します。残りの溶液は淡黄色の上澄み液と分離された色素顆粒を含む赤色のペレットを含むべきです。 </li></ol></li></ol></li></ol> 3.顔料の抽出<ol><li> 酸性メタノール（塩酸-メタノール）の製造 <ol><li>慎重にメタノール5ml（メタノール）に濃縮された（36.5から38までパーセント（w / w）の）塩酸（HCl）の25μlを添加します。スクリューキャップガラス製サンプルバイアル中のHCl-MeOH溶液を保管してください。溶液を均一に混合するために穏やかに渦巻。 注：複数の酸性溶液、より多くの顔料はFiの中に抽出されます最初の抽出。 </li></ol></li><li> 顔料の抽出 <ol><li> 14,000×gで5分間（2.2.2から）顔料顆粒サスペンションを遠心し、廃プラスチックのバケツに上清を捨てます。 </li><li>塩酸 - メタノール溶液500μlを加え、3-4分〜のために各サンプルをボルテックス。 10分（〜40 kHzの）のためのサンプルを超音波処理し、その後、14,000×gで5分間遠心します。 </li><li>トランスファーピペットを用いて、1ドラムスクリュートップガラスバイアルに上清を収集します。上清の色は暗赤色でなければなりません。 </li><li>それ以上の色が抽出されなくなるまで抽出ステップ3.2.2を繰り返し（〜4-5回の洗浄）。 注：各抽出は、顔料の〜1.5ミリリットルを得られます。残りの無色のペレットは、顔料抽出した顆粒です。注：無色顆粒抽出顔料の両方は、さらなる使用まで4℃で水中に保存することができます。 </li></ol></li></ol>抽出プロセス全体を通じて4.キャラクタリゼーション<ol><li> スキャンエル顕微鏡ectron <ol><li>抽出プロセスの純度を確認するために、走査電子顕微鏡（SEM）を用いて（ステップ2.2.1および顔料抽出ステップ3.2.3からの未反応の）画像顆粒。 </li><li>イメージングのための顆粒サンプルを調製するには、最初の遠心分離の未反応の顆粒と顔料の両方が14,000×gで5分間顆粒を抽出し、廃プラスチックのバケツに上清を捨てます。再懸濁これらをエタノール1mlに、転送ピペットを用いてアルミニウムSEMスタブ上に顆粒懸濁液を2-3滴を堆積させます。 </li><li>一晩の試料を乾燥した後、15 nmのコーティングを達成するために3分間金 - 白金コーティングでコートスパッタ。 </li><li>ショットキー・エミッタ及び二次電子検出器をSEMを用いて画像。 5-7 kVのビーム電圧および画像への8と9ミリメートルこれらの材料間のワーキングディスタンスを使用します。 </li></ol></li><li> 紫外-可視（UV-VIS）分光光度測定 <ol><li>吸光度のプロを監視します200〜800ナノメートルからデュアルビーム分光光度計を使用して、すべての3つの条件のためのファイル（2.2.2からの未反応顆粒、3.2.3から抽出された顔料、および3.2.4からの顔料抽出顆粒）。 </li><li>未反応の顆粒および顔料抽出顆粒のための1ミリリットルキュベットに脱イオン水を用いてブランク測定を収集します。両方のサンプルのためのUV-Vis吸収スペクトルを収集します。 </li><li>抽出された顔料のための1ミリリットルキュベット中の酸性メタノール溶液を使用してブランク測定を収集します。サンプル用のUV-Vis吸収スペクトルを収集します。 </li><li>吸光度ピークがその最大相対的な高さに到達する波長を識別することにより、各試料の最大波長を決定します。 </li></ol></li></ol>

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We have nothing to disclose.

私たちは、イカ色素胞の顆粒から色素を抽出する方法を示しました。特に顆粒を標的とすることによって、私たちの目標は、適応着色の媒介におけるそれらの役割を決定することです。この方法は、バルク組織サンプル14または凍結乾燥した皮膚15を使用して、頭足類の顔料を特徴付けるために設計された従来の報告とは異なります。 このプロトコルは、...

このようなイカ、イカ、タコなどの頭足動的にカモフラージュとシグナリングのため、その外観を変更する能力を持っている。1-6この機能は、色素胞として知られている着色された臓器の選択的な面積の拡大によって部分的にサポートされています。4,7-9色素胞されています筋線維によって放射状に固定されているcytoelastic球形嚢の中に閉じ込められたナノ構造色素顆粒のネットワークを含むソフトアクチュエータ。1,3、それらが作動されるように、色素胞は、臓器全体の顆粒を配布提示表面積が500％によって拡大する。3,7このアクションは色素胞の数の上に協調された場合、10,11は 、動物の全体的な着色が変更されます。それは顔料顆粒は、この色の変化に寄与することが知られているが、それらの組成物が不明のままです。私たちは、将来の組成の研究のために適合させることができる色素胞の顔料を分離し、精製するための手順について説明します。 。3,12色素胞含む組織が頭足類から慎重に摘出を通して収穫されます。消化及び均質化処理は、次に、周囲の組織を解離し、色素胞細胞を分離するために使用されます。ナノ構造の顆粒を繰り返し、超音波処理し、遠心分離を用いて、残りの色素胞から単離され、精製されます。精製の際、顔料は酸性メタノール溶液を使用して蝶の羽からの可視色の抽出から適合されたプロセスにおける顆粒から抽出される。13走査電子顕微鏡（SEM）及び分光光度は色素胞顔料が正常に使用して抽出されていることを確認するために使用されていますこのプロセス。 この方法は、cの分子の寄与を調査するために使用される色素胞顆粒の単離を記載します頭足類でoloration。動物全体から12小分子の抽出は、多くの場合、長くて退屈なプロセスになることがあります。ここでの目的は、頭足類でのナノ構造の顆粒から顔料を取得するための効果的かつ容易なプロトコルの将来の研究者を通知することです。

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

色素胞は、Dから解剖されていますpealeii背マントル（ 図1A、1B）。それらが削除されると、色素胞を溶解させ、着色された顆粒（ 図2A、2B） を出て分離するために遠心分離し、洗浄サイクルを使用して精製されます。酸性メタノール溶液（塩酸-メタノール）は、可溶性色素の抽出物および不?...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

イカDoryteuthis pealeii色素胞におけるナノ構造の顆粒から顔料の抽出のためのプロトコルが提示されています。

イカから顔料を抽出する方法<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

a-method-for-extracting-pigments-from-squid-doryteuthis-pealeii

Research

JoVE Journal

Biology

A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K ビュー.  University of New Hampshire. イカDoryteuthis pealeii色素胞におけるナノ構造の顆粒から顔料の抽出のためのプロトコルが提示されています。 

ビデオ: A Method for Extracting Pigments from Squid Doryteuthis pealeii

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

環境学

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

腹部色素沈着の定量化<em&gt;ショウジョウバエmelanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

遺伝学

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

油中水型エマルジョン：疎水性顔料と水溶性クロロフィル結合タンパク質を組み立てるための新しいシステム

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

化学

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

分離してチラコイド脂質リポソームの珪藻峰 Meneghinianaから光捕集アンテナを組み込む

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

生化学

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

転写産物および代謝物からの精製<em&gt;ショウジョウバエ</em&gt;ヘッド

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

生物学

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

植物や緑藻の光捕集複合体のin vitro再構成では、

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

ステージング蛹期間およびショウジョウバエ guttiferaの翼色素沈着測定のためのメソッド

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

ゼブラフィッシュを用いた色素沈着制御因子同定のための逆遺伝学的アプローチ

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

イカから顔料を抽出する方法

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海洋生物の組織からの二次代謝産物のAntipredatory活性を評価するために魚の供給研究所バイオアッセイ

腹部色素沈着の定量化

油中水型エマルジョン：疎水性顔料と水溶性クロロフィル結合タンパク質を組み立てるための新しいシステム

分離してチラコイド脂質リポソームの珪藻峰 Meneghinianaから光捕集アンテナを組み込む

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

転写産物および代謝物からの精製

植物や緑藻の光捕集複合体のin vitro再構成では、

ステージング蛹期間およびショウジョウバエ guttiferaの翼色素沈着測定のためのメソッド

ゼブラフィッシュを用いた色素沈着制御因子同定のための逆遺伝学的アプローチ