Name: a fluorescence-based assay of phospholipid scramblase activity
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

This study was supported by the Velux Stiftung (A.K.M.), NIH grant EY024207 (A.K.M.) and the Austrian Science Fund (FWF) project J3686 (B.P.).

リポソームおよびプロテオリポソー​​ムの調製<ol><li>リポソーム生成<ol><li>ガラスシリンジを使用して、POPCのモル比で52.5モル脂質を得るために、丸底フラスコに（クロロホルム中、25 mg / mlで）および160μlのPOPG（クロロホルム中、25 mg / mlで）1,435μlのPOPCを追加：POPG = 9：1。 </li><li> （何の水浴を溶媒のこのボリュームのために必要とされていない）145回転の回転速度で回転蒸発器を用いて...

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スクランブラーゼ活性アッセイは、オプシンは、リン脂質スクランブラーゼ活性2を持っているかを決定するために、もともと私たちを可能にしました。アッセイはまた、（私たちは、図1AにNBD-PCのために示すように、アシル鎖にNBDで標識されたようなNBD-ホスファチジルエタノールアミンなどのNBD標識化レポーター脂質、さまざまなを使用、または上で私たちは、特異性?...

光受容体ロドプシン、（参照1、例えばレビュー）プロトタイプのGタンパク質共役型受容体は、同定され、生化学的に2,3を検証するための最初のリン脂質スクランブラーゼです。 Scramblasesは、双方向、ATPに依存しない方法4-6で二層間のリン脂質の動きに、生理学的に適切なレベルの本質的に遅い速度を向上させるリン脂質トランスポーターです。彼らの行動の例としては、小胞体および構成スクランブリングは、膜の恒常性と成長のためだけでなく、グリコシル化経路5の様々なために必要とされる細菌の細胞質膜で見つけることができます。規制リン脂質スクランブリングは、それが、マクロファージ7は、「食べる・私」-signalとして作用し、タンパク質因子の産生を触媒する活性化血小板の凝血促進表面を提供する、アポトーシス細胞の表面上のホスファチジルセリン（PS）を露出させるために必要とされます血液凝固に必要なのです。感光ディスク膜に、ロドプシンのスクランブル活性は、ATP依存性により生成される二重層の二つの膜リーフレットの間のリン脂質の不均衡を相殺することが提案されている、一方向性脂質は、ABCA4 4,8,9 10-12フリッパーゼ。 ロドプシンは、原形質膜でのPS露出のために必要2+依存性scramblasesが（参照して見直さ2、TMEM16タンパク質ファミリーのメンバーは、Caとして同定された感光体ディスクでスクランブラーゼとして報告されるまでscramblasesの生理学的重要性にもかかわらず、彼らのアイデンティティはとらえどころのないままでした13）、および細菌タンパク質FTSWは、ペプチドグリカン合成14のために必要な脂質IIスクランブとして提案されました。これらの発見は、方法論のdescribを使用して、得られたプロテオにおけるリポソーム及びスクランブ活動のデモンストレーションで精製されたタンパク質の再構成に基づいていましたここ編。他の潜在的なscramblases 15-21 -ペプチドグリカン生合成に関与MurJとAMJタンパク質、WzxEとO抗原前駆体をスクランブリングに関与し、関連するタンパク質、細菌の細胞質膜を横切ってアミノアシル化ホスファチジルを移行することが必要なMprFタンパク質、および提案されているXkr8ファミリーメンバーアポトーシス細胞の表面上にPSを露出させるためには、 - 生化学的に試験されていません。これは、スクランブ活動を識別し、特徴付けるための堅牢なアッセイの重要性を強調しています。 ここでは、精製されたオプシン、感光体ロドプシンのアポタンパク質、大型単層小胞（LUVを）に、蛍光ベースのアッセイを使用して、得られたプロテオリポソー​​ム内のスクランブ活動のその後の分析の再構成を説明します。いくつかのよく記載されているプロトコルは、したがって、私たちはしません、オプシンの異種発現および精製のための文献で利用可能がありますこのプロトコルでそれを記述。我々は約100ngでFLAGタグ、耐熱性オプシン得ゴーレンら 3に記載されたプロトコルを使用/ 0.1％μL（w / v）のドデシルマルトシド（DDM）。 再構成は、彼らが膨潤するが溶解しないように、十分な洗剤でたLUVを処理することにより達成されます。これらの条件下では、膜タンパク質 - タンパク質 - 界面活性剤ミセルの形態で供給される - は、リポソーム内に統合し、プロテオリポソー​​ムを生じ、界面活性剤の除去時にリポソーム膜に再構成となるであろう。 （0.1％（w / v）のDDMで精製タンパク質として得られる）オプシンを再構成するために、たLUVはPOPC（1-パルミトイル-2- oleoyl- SN -glycero -3-ホスホコリン）とPOPGの混合物（1-から調製されますパルミトイル-2- oleoyl- SN -glycero -3- [ホスホrac-（1-グリセロール）]）とオプシンとNBD-PCを追加する前に、DDMで飽和。界面活性剤は、その後、ポリスチレンビーズとサンプルを処理することによって除去されます。 LASS = &quot;jove_contentは&quot;&gt;蛍光ベースのアッセイの基礎となる原理は、図1Bに示されています。 LUVをは対称的にNBD-PCまたは他のNBD標識蛍光リン脂質レポーター（ 図1A）の微量で再構成されています。ジチオナイトを追加することで、NBDのニトロ基のような膜非透過性ジアニオン、LUVをの外側のリーフレットにおけるNBD-PC分子がレンダリングされる非蛍光は、非蛍光アミノ基に還元されます。 NBD-PC分子もジチオンいずれも実験（&lt;10分）の時間スケールで膜を通過することができるように、これは、蛍光シグナルの50％の減少をもたらします。リポソームをスクランブで再構成されている場合は、内部リーフレットにおいてNBD-PCの分子は、それらが低減されている外部に迅速にスクランブルすることができます。これは、理想的な場合（ 図1C）における蛍光の総損失をもたらします。 エス/ ftp_upload / 54635 / 54635fig1.jpg &quot;/&gt; 図1：スクランブラーゼ活性アッセイの概略図アッセイは、蛍光NBD標識化レポーター脂質を使用しています;。 NBD-PCが示されている（A）。大型単層小胞は、NBD-PCの微量で再構成されています。再構成は、外側と内側のリーフレットに均等に分配NBD-PCで、対称的な小胞を生成します。亜ジチオン酸（S 2 O 4 2-）は 、化学的に非蛍光アミノ基とNBDのニトロ基を低減します。ジチオン酸は、負に帯電し、NBD-PCの分子と反応する膜を通過することはできません：外葉で唯一のNBD-PC分子が低減されるので、ジチオナイト（B、上）とのタンパク質を含まないリポソームの治療は、蛍光の50％の減少を引き起こします内側のリーフレットインチオプシン含有プロテオリポソーム（B、下）、 すなわち 、スクランブラーゼ活性のプロテオリポソーム、Fの100％損失の結果の亜ジチオン治療オプシンとしてluorescenceは、内側と外側のリーフレットの間NBD-PCの移動を容易にします。 （C）は、亜ジチオン酸でタンパク質を含まないリポソームとオプシン含有プロテオリポソームを処理して得られた理想化された蛍光トレースを示します。蛍光損失率は、亜ジチオン酸によるNBDの化学的還元は、スクランブリングは、化学反応の速度以上の速度で発生し、その律速であることを示す両方の場合で同じです。実際の実験から得られたトレースを図3に示す。 <a href="https://www.jove.com/files/ftp_upload/54635/54635fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 我々が説明した方法は、他の精製されたタンパク質、ならびに界面活性剤22とミクロソームを抽出することにより、例えば、得られた膜タンパク質の混合物を再構成し、アッセイするために使用することができます。

<table border="0" cellpadding="0" cellspacing="0" width="1148">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td style="width:141px;">Avanti Polar Lipids</td>
			<td style="width:132px;">850457C</td>
			<td style="width:193px;">POPC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>840457C</td>
			<td>POPG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-an-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>810130C</td>
			<td>NBD-PC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid</td>
			<td>VWR Scientific</td>
			<td>EM-5330</td>
			<td>HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma</td>
			<td>S7653-1KG</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dodecyl-&#946;-d-maltoside&#160;</td>
			<td>Anatrace</td>
			<td>D310 5 GM</td>
			<td>DDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorimeter cuvettes</td>
			<td>sigma</td>
			<td>C0918-100EA</td>
			<td>cuvettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrofluorometer</td>
			<td colspan="2">Photon Technology International, Inc.</td>
			<td>fluorimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydrosulfite technical grade, 85%</td>
			<td>Sigma</td>
			<td>157953-5G</td>
			<td>dithionite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism 5 software</td>
			<td></td>
			<td></td>
			<td>Prism</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>VWR</td>
			<td>JTX171-3</td>
			<td>Tris</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIPEX 10 mL extruder&#160;</td>
			<td>Northern Lipids, Inc.</td>
			<td></td>
			<td>Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman, Drain disc, PE, 25 mm</td>
			<td>Sigma</td>
			<td style="width:132px;">28156-243</td>
			<td>Disc support</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nuclepore Track-Etched Membranes, 0.4 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110607</td>
			<td>400 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:683px;">Whatman Nucleopore Track-Etched Membranes, 0.2 &#181;m, 25 mm diameter</td>
			<td>Sigma</td>
			<td style="width:132px;">WHA110606</td>
			<td>200 nm membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate</td>
			<td>Sigma</td>
			<td>S3264-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VWR Culture Tubes, Disposable, Borosilicate Glass, 13x100 mm</td>
			<td>VWR Scientific</td>
			<td>47729-572</td>
			<td>glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perchloric acid</td>
			<td>Sigma</td>
			<td>30755-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Molybdate Tetrahydrate</td>
			<td>Sigma</td>
			<td>A-7302</td>
			<td>ammonium molybdate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(+)-Sodium L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25G</td>
			<td>sodium ascorbate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bio-Beads SM2 adsorbents</td>
			<td>Bio Rad</td>
			<td>1523920</td>
			<td>polystyrene beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;2.0 mL Microtubes clear</td>
			<td>VWR Scientific</td>
			<td>10011-742</td>
			<td>Reconstitution tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reconstitution glass tube</td>
			<td>VWR Scientific</td>
			<td style="width:132px;">53283-800</td>
			<td>Reconstitution glass tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer&#160;</td>
			<td>Malvern&#160;</td>
			<td></td>
			<td>DLS</td>
		</tr>
	</tbody>
</table>

a fluorescence-based assay of phospholipid scramblase activity

Scramblasesは、ATP非依存的に双方向膜二重層を横切ってリン脂質を移動さ。識別され、生化学的に検証された最初のスクランブラーゼはオプシン、感光体ロドプシンのアポタンパク質でした。ロドプシンは、光の知覚の原因である網膜の桿体ディスク膜に局在するGタンパク質共役受容体です。ロドプシンのスクランブ活動、 すなわち 、そのリガンド11- シス -レチナールに依存しない、アポタンパク質オプシンは、スクランブラーゼとしても活躍。スクランブリング構成および調節リン脂質は、細胞生理学において重要な役割を果たしているが、わずか数リンscramblasesこれまでオプシン他に特定されています。ここでは、オプシンのスクランブラーゼ活性の蛍光ベースのアッセイを記載しています。オプシンは、ホスファチジルコリン、ホスファチジルグリセロール及び蛍光NBD標識PCの微量（1-Pで構成される大単層リポソームに再構成され、almitoyl -2- {6- [7-ニトロ2-1,3ベンゾオキサジアゾール-4-イル）アミノ]ヘキサノイル} - SN -glycero -3-ホスホコリン）。スクランブラーゼ活性は、小胞の内部小葉に位置NBD-PCの分子はその蛍光が化学的に膜を通過することができない還元剤によって除去される外層にアクセスすることができる程度を測定することによって決定されます。我々が説明した方法は、一般的な適用性を有しており、他の膜タンパク質のスクランブ活動を特定し、特徴付けるために使用することができます。

我々は、蛍光ベースのアッセイを使用して、そのスクランブ活動を特徴づけるためたLUVへオプシンの再構成を説明します。私たちは、スクランブルオプシン媒介リン脂質の速度に下限を配置するとオプシンは、機能的に小胞に再構成したオリゴマー状態を決定するために、結果を分析します。 最適な再構成条件を特定す?...

Watch this Scientific Journal Video about A Fluorescence-based Assay of Phospholipid Scramblase Activity at JoVE.com

A Fluorescence-based Assay of Phospholipid Scramblase Activity

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

リン脂質スクランブラーゼ活性の蛍光ベースのアッセイ

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

The overall goal of this procedure is to reconstitute a typical G protein coupled receptor into large unilamellar vesicles, and to measure it's ability to carry out 80p independent translocation of phospholipids across a lipid bilayer using a flurorescence-based assay. This method can help answer key questions in the inter-cellular lipid transport field, such as what is the molecular identity of proteins that can scramble off liplipids and what is their mechanism. The main advantage of this technique is that it is very reliable and robust.Once your reconstitution procedure is established for a given lipid composition and a preferred detergent. To begin this procedure, use a glass syringe to add 1435 microliters of a 25 milligram per milliliter solution of POPC and chloroform to a round bottom flask. Add 160 microliters of a 25 milligram per milliliter solution of POPG and chloroform to obtain 52.5 micro-malipids in a nine to one molar ratio of POPC to POPG.Next, dry the lipids for 30 minutes using a rotary evaporator set to 145 RPM. Once drying is complete, transfer the flask to a vacuum desiccator for three hours at room temperature. Then, add 10 milliliters of buffer A to hydrate the died lipid film.Gently swirl the flask until a homogeneous turbid suspension is formed. Sonicate the suspension at a frequency of 40 kilohertz in a room temperature water bath for 10 minutes. Then, using an extruder, pass the sample through a membrane with a 400 nanometer pore size, 10 times.Follow this with a second cycle of extrusion, passing the sample through a membrane with a 200 nanometer pore size, four times. Set aside the resulting visibly clearer suspension, which contains large unilamellar vesicles of phospolipids about 175 nanometers in diameter. After the extrusion is complete, prepare standards in 13 by 100 millimeter glass tubes as outlined in the text protocol.Then, add 10 microliters of each LUV and proteoliposome sample to a unique glass tube and dilute with 40 microliters of deionized distilled water. Add 300 microliters of perchloric acid to each tube, and heat for one hour at 145 degrees Celsius in a heating block. Place a marble on each tube to prevent evaporation.Then let the tubes cool to room temperature and add one milliliter of deionized distilled water to each. Add 400 microliters of a freshly prepared solution containing 12 grams per liter of ammonium molybdate and 50 grams per liter of sodium ascorbate to each, and vortex to mix. Heat the tubes for 10 minutes at 100 degrees Celsius using marbles to prevent evaporation.Then remove the tubes from the heating block and allow them to cool to room temperature. Using a spectrometer set to 797 nanometers, obtain a calibration curve using the standards. Then, measure the absorbence of the samples against the blank, and determine the phosphate content.Begin by pipetting 800 microliters of each extruded LUV suspension into a unique two milliliter microfuse tube. Add 5.3 microliters of buffer A and 34.7 microliters of 10%mass-per-volume DDM, dissolved in buffer A, to each tube. Next, incubate the samples for three hours at room temperature with end-over-end mixing.While the samples are incubating, weigh out 400 milligrams per sample of beads in a glass beaker. Wash the beads with five milliliters per sample of methanol, stirring slowly for 10 minutes. Repeat this wash once for methanol, three times with water, and once with buffer A.During the last 30 minutes of sample incubation, dry 9.5 microliters-per-sample of NBD labeled phospholipid in a screw-cap glass tube under a stream of nitrogen.Then, add 45 microliters-per-sample of 0.1%mass-per-volume DDM in buffer A to dissolve the dried phospholipid. After incubation is complete, add the dissolved MBD labeled phospholipid solution to each sample. Then, add 0.1%DDM, buffer A, and the DDM-solubilized opsin in the desired amount, in that order, to obtain a final volume of one milliliter with a concentration of seven millimolar DDM.Reconstitution of a membrane protein is achieved by treating vesicles with sufficient detergent so that the swell will not dissolve. And that these conditions, which have to be empirically found for every lipid composition and detergent, the protein will integrate into the liposomes. Mix the samples for one hour at room temperature.Then, add 80 milligrams of the prepared polystyrene beads to each tube, and incubate again for one hour at room temperature with end-over-end mixing. Add an additional 160 milligrams of polystyrene beads to each sample, and incubate for two hours at room temperature with end-over-end mixing. When the incubation is complete, transfer the samples, leaving the beads behind, to clean, glass tubes, each containing 160 milligrams of fresh beads.Mix end-over-end overnight at four degrees Celsius, protected from light. Next, transfer the samples, without the beads, to unique microfuse tubes. Place the tubes on ice in preparation for the scramblase activity assay.Begin by adding 1950 microliters of buffer A to a plastic cubette, containing a plastic stir bar. Then, add 50 microliters of the prepared protealiposome sample. Let the sample equilibrate in the fluorescent spectrometer with constant stirring for five seconds.While the sample is equilibrating, prepare a solution of one molar dithionite in 0.5 molar unbuffered tris. Start the fluorescence monitoring with an excitation of 470 nanometers, an emission of 530 nanometers, and a slit width of 0.5 nanometers. Record for 50 seconds to achieve a stable signal.Then, add 40 microliters of one molar dithionite solution to the cubette. Record the fluorescence for at least 500 seconds. Finally, analyze the data as outlined in the text protocol.For a complete analysis of the fluorescence data, when it's to determine the fraction of vesicles that cannot be reconstituted. The recovery of proteins and lipids in the reconstituted vesicles and also the size distribution of the vesicles. In this study, opsin is reconstituted into large unilamellar vesicles to characterize its scramblase activity using a fluorescence-based assay.Vesicles are reconstituted with NBD-PC, which equally distributes between the outer and inner leaflets. Dithionite reduces the nitro group of NBD on the outside of the vesicle to a non-fluorescent amino groups resulting in a 50%reduction of fluorescence in protein-free liposomes. However, when opsin facilitates NBD movement between the inner and outer leaflet, a complete loss of fluorescence occurs.The representative results of this approach at different protein-to-phospholipid ratios can be seen here. The extend of fluorescence reduction increases with the amount of opsin reconstituted into vesicles, which ranges from zero to 4.95 micrograms. Dithionite was added at the indicated time, and NBD fluorescence was monitored for 400 seconds.Fluorescence reduction is more significant at higher protein-to-phospholipid ratios, with the largest reduction being 85%The endpoint fluorescence reduction is then used to determine the probability of scramblase activity, the likelihood of vesicles having at least one scramblase using Polson statistics. The red curve represents a mono-exponential fit for opsin. While the dashed grey lines represent mono-exponential fits for the reconstitution of opsin into vesicles as monomers, pre-formed dimers, or pre-formed tetramers.The method we describe can easily be adapted to different needs. The procedures are versatile, and can be used to identify and characterize phospholipid scramblase activity of other membrane proteins in the future. The reconstitution procedure can be applied to any membrane protein.This technique enabled us to identify opsin as the first biochemically verified phospholipid scramblase and allowed us, then, to characterize the activity of different confirmation states of the protein, and its ability to transport different lipids.

Research

JoVE Journal

Biochemistry

Expression, Purification, and Antimicrobial Activity of S100A12

S100A12の発現、精製および抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

生化学

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

アクリルアミドゲル電気泳動法で可視化した近赤外蛍光標識 DNA を用いた DNA ポリメラーゼ活性測定法

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

TRNA イソペンテニル トランスフェラーゼ活性を検出する体外の試金

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

ナノ粒子追跡によるひと血中の細胞小胞単一の急速な蛍光特性評価

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

プロテアーゼ活性の画面に蛍光ペプチド胸の谷間アッセイ: アプリケーション コロナ ウイルスのスパイク蛋白質のアクティブ化

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

ショウジョウバエメラノガスターにおけるクエン酸シンターゼ活性の比色アッセイ

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

糸状菌のタップタグ付きヒストンデアセチラーゼのシングルステップエンリッチメント酵素活性アッセイのためのカビnidulans

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

核酸プローブを用いたヌクレアーゼ活性の運動スクリーニング

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

ホスファチジルセリン/ホスファチジルイノシトール4-膜間のリン酸交換の蛍光ベースの測定

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...