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1。脂質の抽出<ol><li>寒天培地上で生育した植物から30mgを4週齢のシロイヌナズナの葉を収穫によって脂質の抽出を開始する培地や土壌を固化し、1.5 mLのポリプロピレン反応チューブに移す。新鮮な葉は、フラッシュは、液体窒素中で凍結し、-80℃で保存することができます</li><li>各サンプルにメタノールから成る溶剤300μL抽出、クロロホルムとギ酸（午前20時10分01秒、V / V / V）を追加します。 5分間（ペイントシェーカーまたは同様のを使用して）を激しく振る。 </li><li> 0.2 Mリン酸（H 3 PO 4）、1 M塩化カリウム（KCl）と渦簡単に150μLを加える。 </li><li> 1分間、室温で13000 × gで遠心する。下のクロロホルム相に溶解した脂質は、TLCプレート上にスポットされます。 </li></ol> 2。薄層クロマトグラフィー（TLC）15 <ol><li>水没0.15 Mの硫酸アンモニウム（（NH 4）2 SO 4）溶液中に30秒のためにストリップを読み込むと20cmx20cmシリカゲルコーティングTLCプレートを、TLCプレートを準備するには、30秒間水没した後、中に少なくとも2日間、プレートを乾燥させる対象となるコンテナ。アクティベーション中にアンモニウムの昇華は、他のグリセロ脂質からの分離のために必要なホスファチジルグリセロールをプロトン化硫酸、背後に残します。 </li><li>実験日に、アクティブには、120℃のオーブンで焼成することによりプレートをTLC 2.5時間° C。 </li><li>室温に活性化プレートを冷却した後、クロマトグラムの原点にプレートを介して直線（プレートの端から1.5 cm）を描画するために鉛筆を使用してください。 </li><li>ドラフトでは、ゆっくりとN 2の遅い流れの下、200μLの黄色いプラスチックのヒントと20μLピペットを使用して、より低いクロロホルム相の脂質抽出物の3 X 20μLを提供します。この目的のために、タイゴンチューブは、N 2タンクのレギュレータに接続されている。直径1cm未満のスポットを保持します。各プレートは、10サンプル（その後のGLC分析が計画された時点）を保持することができます。 </li><li>脂質は、ドラフト内で完全に乾燥したスポットとして、アセトン、トルエン、水（30 mLを7.5 mLの91 mL）で構成される展開溶媒を準備。周囲の相対湿度が高い場合、分離に影響を及ぼす可能性があります。所望の分離を達成するためにこのケースでは水は（7.0 mLの：：30 mLの91 mL）を与えるために軽減する必要があります。 </li><li>と下に向けて、サンプルの端にタンクにプレートを置く室（：H：W = 27.0：：26.5 7.0、CM / CM / CM L）を開発する密閉式TLCに展開溶媒80mlを注ぐ。クランプを使用してタンクを密閉する。溶剤は、プレートを登っれると脂質が分離されます。開発時間が室温で約50分です。 </li><li>溶剤フロントが板の上から1 cmに達したときに、慎重にタンクからプレートを取り外し、約10分間、ドラフト内で完全に乾かしてください。 </li><li> TLCで分離された脂質は、いずれの可逆的定量分析のためにヨウ素で簡単に染色または不可逆的に硫酸またはα-ナフトールで染色することができます。 <ol><li>硫酸炭化：120ヒュームフードと焼くのスプレーガラスのスプレーボトルの水の50％硫酸でプレート℃で15分間（図2A）。 </li><li>糖脂質のためのα-ナフトール染色：120 ° Cで2.4％（w / v）の10％のα-ナフトール（v / v）の硫酸、80％（v / v）エタノールと焼くとプレートをスプレー3-5糖脂質のバンドまでの分は、（図2B）、ピンクや紫色に染色される。過剰治療は、試薬中の硫酸の存在のためにすべての脂質の焦げにつながります。 </li><li>ヨウ素染色：ドラフト内で、ヨウ素の結晶を持つ閉じたTLCタンク（脂質が見えるようになるまでヨウ素蒸気を大気の飽和に至る下部にあるトレイの中）にプレートを置く。長すぎるヨウ素は、共有結合多価不飽和脂肪酸（図2C）を変更する可能性があるため、ヨウ素にプレートを公開しないでください。また、脂質のいずれかの酸化を避けるために、サンプルのレーンに混在のみ標準の車線は、ガラスウールを使用して染色されるべきであるN 2は、個々の標準車線以上の吹くに使用しているヨウ素の結晶をパスツールピペットを差し込ん。 </li></ol></li></ol> 3。脂肪酸アシルメチルエステル（FAME）反応16 <ol><li>かみそりの刃を持つTLCプレートから同定された脂質のスポットを囲むシリカを削除します。脂質含有シリカをこすり取ってテフロン（PTFE）ライニングスクリューキャップ付きガラス管に漏斗を用いてシリカ粉末を転送する。 </li><li>ガラスピペットで各サンプルに無水メタノールで1mLの1N塩酸（HCl）を追加します。 </li><li> 200μLの黄色いプラスチックチップで200μLピペットを用いて内部標準として、各サンプルに200μLピペットを用いて100μL50μgのmL -1のペンタデカン酸（15:0）を追加します。管コントロールとしてメタノール性塩酸でのみpentadecenoic酸としてください。テフロンライニングキャップでしっかりとガラス管を閉じます。 </li><li>インキュベートGL25分間80℃の水浴中でお尻のチューブ。チューブは、溶媒が蒸発しないように密封する必要があります。 </li><li>チューブが冷却した後、精力的に1mLのヘキサンと渦が続く1 mLの0.9％塩化ナトリウムを追加。 3分間1000xgでサンプルを遠心分離します。 </li><li>ドラフトでは、パスツールピペットで試料のヘキサン/上位レイヤを削除し、新しい13x100 mmのガラス管に入れてください。 </li><li>完全に乾燥させることなく、N 2の遅い流れの下でヘキサンを蒸発させる。 </li><li> 60μLヘキサンで生じる脂肪酸アシルメチルエステルのを溶かす。しっかりとオートサンプラバイアルとキャップにサンプルを移す。サンプルは、° C、短期的、-20℃で数日間にて保存することができます。 </li></ol> 4。気-液クロマトグラフィー（GLC）10 <ol><li> GLCを開始する前に、ヘリウム、水素と空気ボンベが満たされていることを確認します。 </li><li>十分なヘキサンを溶媒リザーバーに追加する必要があり、廃棄物の容器を空にする必要があります。脂肪酸アシルメチルエステルの分離のために、マシンにDB - 23カラムを取り付けます。 </li><li>オートサンプラにバイアルを置きます。システムのコンピュータ上でGLC用ケミステーションソフトウェアを起動します。 </li><li> 250℃ヘリウム流量の48.6 mLの分-1の速度と21.93 psiで圧力とC。で入口温度を設定するスプリット比は30.0である：1。 </li><li>オーブンの温度を2分間140℃で最初に設定し、° C 25 ° C min -1の速度で160に送出されます。 -その後160から増加させる° C〜250 ° C ° C min -1の 8の割合で、250で保持℃〜140減少に続いて4分間C ° C 38の速度で℃以上の温度を設定する1。一度の実行では、約21分かかります。 </li><li>水素炎イオン化検出器の温度は270℃です30.0 mLの分-1、30.0 mLの分-1で400 mLの分-1とヘリウムの流量で空気流量の水素流量とC。 </li><li>バイアルおよび実行シーケンステーブルにサンプル名の数を入力してください。バイアルあたり2μLのサンプルを注入して10μL注入器を設定します。 </li><li>楽器の準備が整うと、実行シーケンスを開始する。 </li></ol> 5。代表的な結果： 4週齢のシロイヌナズナの苗からTLCで区切られた脂質の不可逆的な染色の例を図2に示されています。硫酸ステンド脂質（図2A）は黒焦げと褐色の斑点として表示されます。 α-ナフトールは、他の極性脂質が（図2B）、黄色の染色ながら、α-ナフトールはピンク紫色を運ぶで染色等の糖脂質をSQDG、そのようなMGDG、DGDGとして糖脂質を染色することが好ましい。ヨード染色では、可逆的であると脂質のヨウ素が蒸発する（図2C）のような短い時間の経過とともに消え黄色がかった色を与えます。未染色の脂質は脂質の分解を低減することが望ましいですが一時的にヨウ素染色された脂質をGLC分析に供することができる。 が正常に完了した場合、異なる脂肪酸アシルメチルエステルを表す特徴的な信号は、GLC（図3）後に観察されます。短い炭素鎖と少ない二重結合を有する脂肪酸アシルメチルエステルは、DB - 23カラムを使用してより短い保持時間を持っている。脂肪酸アシルメチルエステルのプロファイリングは、変更された脂質組成を有する変異体を識別するための敏感なツールです。図4では、MGDG18：3脂肪酸のモル比が野生型18に比べてtgd4 - 1変異体で減少している。すべての脂質クラスのモルワン脂質クラスの脂肪酸アシルメチルエステルのモル数を分割することによって、それぞれの脂質のモル比が計算されます。たとえば、MGDGのモル比を計算する： （MGDG）モル％=Σ[のFAME（MGDG）] /Σ[のFAME（合計）] × 100％ 野生型と変異型の両方からの各脂質クラスの結果のモル比を比較することができます。例えば、tgd4 - 1変異体は、MGDGとPGの相対量を増加させたがDGDGとPE（図5）18の量を減少している。 <img src="/files/ftp_upload/2518/2518fig1.jpg" alt="図1" /> シロイヌナズナ実生を用いて極性脂質の分析の図1。フローチャート。総脂質は、4週齢のシロイヌナズナの苗から抽出し、TLCによって分離される。分離された脂質をGLC分析に続いてエステル交換のためのTLCプレートから掻きすることができます。 <img src="/files/ftp_upload/2518/2518fig2.jpg" alt="図2" /> 図2。TLCプレート上に脂質の分離。 35 mgの脂質抽出物（新鮮重量）は、野生型の実生は、TLCにより分離し、硫酸（）、α-ナフトール（B）またはヨウ素蒸気（C）で染色されています。三繰り返しは、それぞれの染色法で示されています。 DGDG、digalactosyldiacylglycerol、MGDG、monogalactosyldiacylglycerol、PC、ホスファチジルコリン、PE、ホスファチジルエタノールアミン、PG、phosphatidylglyceROL、PI、ホスファチジルイノシトール、SQDG、スルホキノボシルジアシルグリセロール。 <img src="/files/ftp_upload/2518/2518fig3.jpg" alt="図3" /> 野生型のMGDGから派生した脂肪酸メチルエステル（FAME）の図3。GLC分析。のFAMEが30メートルキャピラリーカラムで分離し、水素炎イオン化によって検出されます。ペンタデカン酸（15:0）は内部標準として使用されます。 <img src="/files/ftp_upload/2518/2518fig4.jpg" alt="図4" /> 図4。野生型Col2に（白のカラム）とtgd4 - 1変異体（黒色カラム）でMGDGの脂肪酸プロフィール。脂肪酸は、二重結合の数が続く炭素の数として表現さ​​れています。三繰り返しは平均され、標準偏差が表示されます。 <img src="/files/ftp_upload/2518/2518fig5.jpg" alt="図5" /> 図5。野生型Col2に（白のカラム）の極性脂質の組成とtgd4 - 1変異体（黒色カラム）。三繰り返しは平均され、標準偏差をエラーバーで示されている。

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GLCと相まってTLCは、植物の極性脂質の定量分析のための堅牢かつ迅速なツールを提供しています。脂質組成のわずかな変化を識別することができるので、この方法は、極性脂質の代謝経路1,20で損なわ変異の大規模スクリーニングのために使用されています。この方法は、広く基質として極性脂質を利用する酵素の活動を監視するために使用されます。2,6,7 葉のほかに、そのような根や種子や葉緑体やミトコンドリアなどの細胞内の画のような他の植物組織の脂質組成物はまた、同じ方法で決定することができます。 ここで使用される溶媒系（アセトン、トルエン、水）は、植物の糖脂質とリン脂質の分離に最適化されています。 tetragalactosyldiacylglycerolがPCで実行中しかし、tgd1、2,3,4変異体と分離された葉緑体で、TGDGはPEと一緒に動作します。この場合、クロロホルム、メタノール、酢酸および水（85：20：10：4、V / V / v / v）を溶媒系は​​13を使用されています。時々 、二つの異なる溶媒系を用いて二次元TLCは、さらに別の糖脂質とリン脂質19に実行されます。さらに、植物組織は、直接TLC 5の初期分離することなく、総脂肪酸のプロファイルを決定するためにGLCが続くFAMEの反応に供することができる。実証TLC - GLCのシステムの横に、脂質プロファイリングに使用する別の方法は、直接エレクトロスプレーイオン化タンデム質量分析法17に基づいています。この方法では抽出物中の脂質の初期のクロマトグラフィー分離は省略されます。しかし、この方法では、ラボや変異体スクリーニングのためのルーチン分析のためのそれはさほど便利で高価な設備と経験豊富な人材を、必要になります。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)

環境からの生物学的膜を別のセル。このようなホスファチジルコリンまたはホスファチジルエタノールアミン、細胞とその周囲との間の化学交換のための両方の境界とのインタフェースとして機能するフォームの二分子膜として多細胞の植物や動物、グリセロ脂質、単一の細胞から。動物とは異なり、植物細胞は光合成のための特別な細胞小器官、葉緑体を持っている。 monogalactosyldiacylglycerol（MGDG）、digalactosyldiacylglycerol（DGDG）、スルホキノボシルジアシルグリセロール（SQDG）4：葉緑体の複雑な膜システムは、すなわちユニークなグリセロ脂質、リンを欠く糖脂質が含まれています。これらの脂質の役割は、単に構造を超えています。これらの糖脂質と他のグリセロ脂質は光合成8,11におけるグリセロ脂質の関与を示す光化学系IとIIの結晶構造で発見された。リン酸飢餓時には、DGDGは、リン脂質9,12の損失を補うためにextraplastidic膜に転送されます。 多くのこれらの脂質の生合成と機能の我々の知る限りは、 シロイヌナズナの 14に遺伝学的および生化学的研究の組み合わせから導出されています。これらの調査の間に、極性脂質の分析のための簡単​​な手順では、脂質の変異体のスクリーニングと解析のために不可欠になっており、詳細に概説する。葉の脂質抽出物は、最初の薄層クロマトグラフィー（TLC）によって分離され、グリセロ脂質は、ヨウ素蒸気に可逆的に染色されています。個々の脂質は、TLCプレートから掻きと水素炎イオン化検出器（FID - GLC）（図1）と結合した気 - 液クロマトグラフィーによって分析されている脂肪酸アシルメチルエステル（のFAME）、に変換されます。このメソッドは、変異体スクリーニングのための信頼できるツールであることが証明されています。たとえば、tgd1、2,3,4小胞体からプラスチド脂質輸送変異体は、異常なgalactoglycerolipidの蓄積に基づいて発見されました。trigalactosyldiacylglycerol（TGDG）と18時03分の相対量の減少（炭素：ダブル債）膜脂質3,13,18,20の脂肪酸アシル基。また、このメソッドは、基板6として脂質を用いてタンパク質の酵素活性を決定するための適用可能です。

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

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

極性脂質抽出物の組成と個々のグリセロ脂質の脂肪酸組成は、シンプルで堅牢な脂質プロファイリング実験で決定されます。この目的のために、グリセロ脂質は薄層クロマトグラフィーによって分離されているとそのアシル基のメチル基移転を行った。脂肪酸アシルメチルエステルをガス - 液体クロマトグラフィーによって定量化されています。

気-液クロマトグラフィー（GLC）とあいまって薄層クロマトグラフィー（TLC）によりシロイヌナズナポーラーグリセロ脂質プロファイリング

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

arabidopsis-thaliana-polar-glycerolipid-profiling-thin-layer

Universidad San Sebastian

Research

JoVE Journal

Biology

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

36.6K ビュー.  Michigan State University. 極性脂質抽出物の組成と個々のグリセロ脂質の脂肪酸組成は、シンプルで堅牢な脂質プロファイリング実験で決定されます。この目的のために、グリセロ脂質は薄層クロマトグラフィーによって分離されているとそのアシル基のメチル基移転を行った。脂肪酸アシルメチルエステルをガス - 液体クロマトグラフィーによって定量化されています。

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDIサンプル調製：超薄層法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

生物学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

の14日齢の苗の組織からのプロトプラストの単離シロイヌナズナ</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

ルシフェラーゼ免疫系による抗体のプロファイリング（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

不安定な多タンパク質複合体の単離で in vivoで制御セルラー架橋及び免疫磁気アフィニティークロマトグラフィー

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

フローラルディップ変換のシロイヌナズナ調べに pTSO2：：β-グルクロニダーゼレポーター遺伝子発現

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

タンパク質の精製で使用するための自​​動化された疎水性相互作用クロマトグラフィーカラムの選択

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

から初期段階の胚の効率的かつ迅速な単離<em&gt;シロイヌナズナ</em&gt;種子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

の組織化学的染色<em&gt;シロイヌナズナ</em&gt;二次細胞壁の要素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...