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.).

1.脂质体和蛋白脂质体的制备<ol><li>脂质体形成<ol><li>用玻璃注射器，添加1435微升POPC（25毫克/毫升，在氯仿中）和160微升POPG（25毫克/毫升，在氯仿中）到一个圆底烧瓶中，以获得在POPC的摩尔比52.5微摩尔脂质：POPG = 9：1。 </li><li>使用旋转蒸发器在145 rpm的转速（需要此体积的溶剂没有水浴）干燥30分钟的脂质，然后将烧瓶转移到真空干燥器为至少3小时，或过夜，在室温下（RT）。 </li><li>水合干燥...

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该scramblase活性测定，使我们原来确定的视蛋白具有磷脂scramblase活动2。该测定法还允许我们通过测试特异性表征视蛋白的scramblase活性（我们采用了各种NBD标记的报告脂质如NBD-磷脂酰乙醇胺，如图所示为NBD-PC在图1A中，或在上的酰基链与NBD标记的首基，NBD-鞘磷脂或NBD-磷脂2），囊泡脂质组合物（的效应， 例如 ，在不同量的作为囊泡组分胆固醇），和是否视蛋白?...

感光体视紫红质，一个典型的G蛋白偶联受体（参考文献1中综述例如），要被识别的第一磷脂scramblase和生物化学验证2,3。 Scramblases是磷脂转运会增加transbilayer磷脂运动的本质慢的速度在双向生理上适当水平，ATP无关的方式4-6。他们的行动的实例可于内质网和细菌胞质膜，其中需要对膜稳态和生长构加扰，以及针对各种糖基化途径5中找到。扰码是需要调节磷脂以暴露凋亡细胞的表面上的磷脂酰丝氨酸（PS），其中它充当一个“吃我” -信号为巨噬细胞7和对活化血小板提供一种促凝表面以催化生产蛋白质因子的š所需血液凝固。在感光体光盘的膜，视紫红质的扰频活动已经提出以抵消由ATP依赖，单向脂质产生的双层的两个薄膜小叶之间的磷脂失调翻转酶ABCA4 4,8,9 10-12。 尽管scramblases的生理重要性，它们的身份仍然难以捉摸直到视紫红质被报告为在感光体圆盘一个scramblase 2，TMEM16蛋白家族的成员被确定为钙离子需要在质膜PS暴露依赖性scramblases（在参考审查13），并作为脂类II scramblase所需肽合成14提出的细菌蛋白质FtsW。这些发现是基于纯化的蛋白质的脂质体中使用的方法describ重构和scramblase活性示范在所得脂蛋白体这里编。其他潜在scramblases 15-21 -的MurJ和AMJ蛋白肽聚糖的生物合成牵连，WzxE和相关蛋白牵涉于扰O-抗原的前体，MPRF蛋白需要穿过细菌胞质膜易位氨基酰化磷脂，并且已经提出Xkr8家族成员以暴露的PS凋亡细胞的表面上 - 有待生化测试。这凸显了一个强大的分析的重要性，以确定和识别scramblase活动。 这里，我们描述了纯化的视蛋白的在使用基于荧光的测定法所得到的蛋白脂质体的重建，感光体的视紫红质的脱辅基蛋白，成大单层囊泡（的LUV）和scramblase活性随后的分析。有在文献中用于异源表达和视蛋白的纯化可用几个充分描述的协议，因此，我们将不描述它在这个协议;我们使用这产生FLAG标签，热稳定视蛋白在约100纳克在戈伦等 3所述的协议/微升的0.1％（重量/体积）十二烷基麦芽糖（DDM）。 重构是通过使得它们溶胀但不溶于治疗与足够洗涤剂的LUV实现。在这些条件下，膜蛋白 - 蛋白质 - 去污剂胶束的形式提供 - 将整合入脂质体和成为重建成在洗涤剂除去脂质体膜，导致脂蛋白体。重构视蛋白（作为纯化蛋白得到的0.1％（重量/体积），DDM），的LUV由（1- POPC（1-棕榈酰-2-油酰- SN -glycero -3-磷酸胆碱）和POPG的混合物制备棕榈酰-2-油酰- SN -glycero -3- [磷酸外消旋- （1-甘油）]）和添加视蛋白和NBD-PC之前，用DDM饱和。洗涤剂然后通过用聚苯乙烯珠粒的样品中除去。 小姑娘=“jove_content”&gt;的基于荧光的测定法的基本原理示于图1B。的LUV对称用NBD-PC或其他NBD标记的荧光磷脂记者（ 图1A）的微量重构。上加入连二亚硫酸盐，膜不通透性二价阴离子，在的LUV的外部小叶NBD-PC分子呈现非荧光作为硝基组NBD的被还原成非荧光氨基 - 基团。既不NBD-PC分子也不连二是能够在时间尺度的实验（&lt;10分钟）的遍历膜，这导致荧光信号的减少50％。然而，如果脂质体与scramblase重构，在内小叶NBD-PC分子可以迅速加扰向外部在那里它们被降低。这导致荧光的在理想的情况下（ 图1C）的总损耗。 ES / ftp_upload / 54635 / 54635fig1.jpg“/&gt; 图1：scramblase活性测定的示意图的测定使用荧光NBD标记的报告脂质; NBD-PC如图（A）。大单层囊泡重构与NBD-PC的痕量。重组产生对称囊泡，以在外部和内部小叶均匀分布NBD-PC。连二亚硫酸盐（S 2 O 4 2-）化学还原硝基组NBD到一个非荧光氨基-基团。用连二亚硫酸盐（B，上图）无蛋白质的脂质体处理引起，因为只有在该外部小叶的NBD-PC分子荧光的减少50％的降低：连二亚硫酸盐被带负电荷的，并且不能穿过膜与NBD-PC分子反应在内部小叶。含有视蛋白，脂蛋白（B，下），连二亚的治疗即 scramblase活性脂蛋白，导致的F 100％的损失luorescence如视蛋白促进内层和外层小叶之间的NBD-PC的移动。 （C）表示对治疗无蛋白质的脂质体和含有视蛋白-蛋白脂质体与连二亚得到理想化荧光痕迹。荧光损失率是在两种情况下，指示由连二亚化学还原NBD的是限速，并且加扰出现在比化学反应的速率等于或大于一个速率是相同的。从实际实验中获得痕迹如图3所示。 <a href="https://www.jove.com/files/ftp_upload/54635/54635fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 我们描述的方法可以被用来重建和测定等纯化的蛋白质，以及获得，例如，通过用洗涤剂22提取微粒的膜蛋白的混合物。

<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易位穿过膜双层磷脂双向在一个ATP无关的方式。第一scramblase被识别和生化核实被视蛋白，感光体的视紫红质的脱辅基蛋白。视紫红质是在它负责的光的感知的视网膜视杆圆盘膜局部的G蛋白偶合受体。视紫红质的scramblase活动并不取决于其配体11- 顺 -retinal， 即载脂蛋白视蛋白还积极为scramblase。虽然组成型和调节磷脂扰在细胞生理学中起重要作用，只有少数磷脂scramblases迄今已除了视蛋白鉴定。这里，我们描述视蛋白的scramblase活性的基于荧光的测定法。视蛋白被重组到磷脂酰胆碱，磷脂酰甘油和荧光NBD标记的PC（1-p的微量组成的大单层脂质体almitoyl -2- {6- [7-硝基2-1,3苯并恶-4-基）氨基]己酰基} - SN -glycero -3-磷酸胆碱）。 Scramblase活性通过测量到位于囊泡的内小叶NBD-PC分子能够访问外部小叶，他们的荧光化学由不能越过膜还原剂消除的程度来确定。我们描述的方法具有普遍的适用性，并可以用于鉴定和表征其他膜蛋白的scramblase活动。

我们描述视蛋白的重建成的LUV使用基于荧光的测定法来表征其scramblase活性。我们分析结果，以放置在视蛋白介导的磷脂加扰的速率的下限，并确定在哪个视蛋白功能上重新构造成囊泡的寡聚状态。 以确定最佳的重组的条件下，有必要确定凭经验必须使用溶胀的LUV使它们接受蛋白质插入洗涤剂的量。这样的实验是在图2A</s...

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.

磷脂Scramblase活动的基于荧光的测定法

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的表达，纯化和抗菌活性

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 ...

科研

生物化学

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

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

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

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

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

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

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

基于荧光的人体血液单细胞外囊快速表征

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

一种用于蛋白质溶解活性的荧光肽裂解检测: 冠状病毒穗蛋白活化的应用

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

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

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

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

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

使用核酸探针进行核酸活性的动力学筛选

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

基于荧光的测量磷酸酯/磷酸二醇 4-磷酸盐膜之间的交换

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