这项工作是由美国国立卫生研究院授予＃DA15169到下摆

程序概述： 使用这种方法，细胞表面的蛋白质共价键上标有使用贩运的限制性条件（即低温）下一个膜impermeant，二硫化碳耦合生物素试剂（磺酸基- NHS - SS -生物素）外赖氨酸残基与生物素（见图1插图）。一个细胞转移到贩卖宽松的条件下（37℃）和生物素化蛋白的内化。其他细胞保持在较低的温度控制为1）总时间= 0的表面蛋白，和2）剥离控制。一化后的短期内，细胞移回低温停止内部化，任何残留的表面生物素是治疗还原剂，劈开二硫键再加生物素细胞剥离。来自细胞表面和内在的生物素标记的蛋白质是保护剥离一步，将是唯一保持生物素化蛋白。细胞裂解，生物素蛋白链霉亲和素亲和层析柱分离和利益的蛋白质是由定量免疫印迹检测。要确定吞率，内在蛋白量相比，总面控制在时间= 0标记。我们已经成功地使用这种方法来测量内部的神经元的去甲肾上腺素 1和多巴胺 1-4转运率。 详细的协议： 第1天： <ol><li>板细胞在6孔板等，他们将第2天〜80％融合。另外，如果正在使用的转染细胞，转染密度的内化率将测得的时间，他们将在〜80％融合。如果没有强烈的贴壁细胞，组织培养器皿应被视为与细胞粘附底物（如聚- D -赖氨酸）广泛的洗涤步骤，以防止在细胞丢失。 </li><li>每个蛋白质正在测试中，板2上的一个板块水井，作为总的表面蛋白（T = 0）和剥离控制。在第二个板块，板块之一为每个被测试的内化条件（即基底吞率与药物治疗）。 </li><li>准备以下解决方案，在第2天使用指定的温度和存储： <ul><li> PBS洗涤2 +：磷酸盐缓冲液（pH值7.4），1.5毫米0.2 毫米氯化钙， 氯化镁 2，（4 ° C）补充</li><li> 生物素淬火解决方案 ：PBS 2 +辅以甘氨酸，100毫米（4℃） </li><li> NT缓冲区 ：150毫米氯化钠，1.0 mM的EDTA，0.2％BSA，20毫米三，pH值8.6，（4℃） </li><li> RIPA缓冲液 ：氯化钠150毫米，10毫米的Tris，pH值7.4，1.0 mM的EDTA，0.1％SDS，1.0％TRITON X 100，脱氧胆酸钠，1.0％（4 ° C） </li><li> 磺酸基- NHS - SS -生物素储备液 ：溶解于二甲基亚砜（DMSO）为200毫克/毫升（-20 ° C） </li><li> 三（2 -羧乙基）膦盐酸盐（TCEP）原液：500 毫米 ，H 2 O（-20 ° C，在铝箔覆盖挡光） </li></ul></li></ol>第2天： <ol><li>准备PBS洗涤2 +辅以0.18g/ml葡萄糖，0.2％，IgG /蛋白酶的无牛血清白蛋白（PBS洗涤2 + /克/ BSA） 。预温至37 ° C水浴中的解决方案。 </li><li>台式磺酸基- NHS - SS -生物素原液解冻融化的二甲基亚砜。立即使用前，准备新鲜的磺酸基- NHS - SS -生物素的溶液（2.5毫克/毫升，在冰冷的 PBS 2 +，足以为0.75毫升/井）。旋涡的解决方案，大力溶解在二甲基亚砜。需要注意的是NHS -生物素试剂在水溶液中很容易水解。因此，所有的解决方案，应立即准备前使用。 </li><li> 生物素：在寒冷的房间里的冰浴上放置板和冲洗用冰冷的 PBS 2 3 × 2毫升。某些板块略有角度，以便完成排水和清除缓冲液。添加0.75 /毫升的新鲜磺酸基- NHS - SS -生物素的解决方案，每个以及。孵育x 15&#39;，4℃冰浴剧烈摇晃彗星。孵化完成后，准​​备另一个新的磺酸基- NHS - SS -生物素的解决方案。更换旧的解决方案，与新鲜的解决方案和孵化× 15&#39;，4 ° C。 </li><li> 淬火：至关重要的是，NHS -生物素分子的所有非反应淬，所以，他们不会做出反应，并biotinylate细胞内的蛋白质，一旦细胞裂解。淬火液洗涤细胞3 × 2毫升和淬火的解决方案2毫升× 15&#39;，4 °与轻轻摇动孵化两次。 </li><li> 内在：如果正在测试的药物治疗，适当的药物浓度， 以PBS 2 + /克/ BSA。保持在4 ° C的控制盘和内化板的冷室。预热PBS洗3 × 2毫升+ / G / BSA（+ / - DRUGS），并留在相同的解决方案（2毫升/孔）。填写任何空井预热的解决方案，以确保整个板块的温度均匀。细胞转移到37℃培养箱中培养10&#39;。立即结束前37 ° C孵化，准备在NT缓冲新鲜的50毫米TCEP解决方案，被剥离的步骤，并存储在冰上。准备足够量为1.0毫升/。 </li><li> 脱模：立即传送板（S），冰浴，并返回到冷房。快速洗细胞与冰冷NT缓冲区，3 × 2毫升停止内吞作用。还用NT的缓冲地带的控制井3 × 2毫升。添加1.0毫升新鲜剥离的解决方案，每孔。在冰上孵育，轻轻摇动15&#39;，4 ° C。井更换新鲜剥离的解决方案，并在冰上孵育15&#39;，4 ° C。 </li><li> 裂解：清洗所有暴露剥离的解决方案，3 × 2毫升NT缓冲井。然后用3 × 2毫升PBS +所有的水井（包括总控制） 。裂解在300μl/wellRIPA缓冲液，含有新鲜的蛋白酶抑制剂（PMSF，1.0毫米1.0微克/毫升每亮肽素，抑肽酶和胃酶抑素）（或其他感兴趣的蛋白质裂解液兼容）。裂解通过晃动× 20&#39;，4 ° C。转移到离心管和清除细胞碎片，离心18000 XG，10&#39;，4 ° C。 </li><li> 测定蛋白浓度：与您的裂解条件（ 例如 DC蛋白测定，Bio - Rad公司）兼容使用蛋白检测，以确定的裂解液中的蛋白浓度相比，一个标准的牛血清白蛋白曲线。 </li><li> 链霉亲和素亲和层析法 ：准备与蛋白质的等值金额，每个样品的离心管。加入裂解缓冲液中，使每个样品的最终体积200μL。旋涡链霉亲和素琼脂糖珠大力带来更被停牌。使用提示200μL的移液器切断，每个管的吸液管珠。推荐微克裂解液20μLbeads/50。在管肩孵育过夜，于4℃。 </li></ol>第3天： <ol><li> 珠清洗：离心机样品，18000 XG 2“收集珠子。吸过裂解液，小心不要接近吸气珠颗粒。更好地控制抽吸结束使用塑料枪头。添加0.75毫升每管细胞裂解液，和涡洗珠。离心样品，18000 XG 2“收集珠子和重复的愿望和洗两次（共三个清洗）。最后一次洗涤后，吸出，而不会干扰磁珠颗粒尽可能多的缓冲区。小费向管抽吸助攻这一步。 </li><li> 样品洗脱：每管加入20 -25μl2X Laemmli样品缓冲液（减少）。还原剂将切割NHS的SS -生物素的二硫键，释放到溶液中分离蛋白质。大多数膜蛋白是非常脆弱的聚合煮沸时，不应该加热前进行SDS - PAGE凝胶装载。确定你感兴趣的蛋白质的热敏感性前进行这些实验。如果蛋白质无法容忍沸腾/加热，旋转器，30&#39;，室温孵育前经SDS - PAGE分析。这是足够的切割二硫键和洗脱的蛋白质。 </li><li> SDS - PAGE电泳和免疫印迹：经SDS - PAGE分离蛋白质。对于每个样本，这是最简单的运行样品，按下列顺序：（时间= 0）的总表面积，带钢，内化的条件1，条件2＃等免疫印迹膜转移与相应的抗体印迹的蛋白质利益。捕获使用CCD相机凝胶的文件系统，一定有不饱和像素的免疫反应乐队。使用凝胶电泳分析/密度测量软件，量化每个波段的密度。 </li></ol>代表性的成果： 一个代表性的免疫印迹结果显示在图2A。信号最强的是“总”巷（T）的，这是表面蛋白总量前的内化。最好应接近空白，这表明，带钢实验的有效的“脱衣”控制（S）。地带效率的计算方法是比较“地带”里的“总”里（如蛋白质，是平行的地带生物素的密度，但既不是加热至37 ° C，也没有暴露剥离的解决方案）。使用下列公式： [1 - （条/总）] × 100％ 使用这个公式，在图2的地带是99.8％的效率。最后，你会看到比（S）（一）在化巷总强度较小的波段。在这个例子中（图2），细胞治疗，治疗无论是车辆或1μm的佛波醇酯（PMA），在10“内化和多巴胺转运的内化率分别测定10”最初的内部化时期。内化率的计算公式为如下： 内在/总* 100 如图2b所示，10.4％的表面的DAT内超过10“车辆处理的条件下。 PMA的治疗增加了23.2％的总表面的DAT DAT内化率。 <img src="/files/ftp_upload/1669/1669fig1.jpg" alt="图1" /> 图1。议定书“的插图。细胞是生物素在4 ° C至专门标签表面的人口，并转移至37 ° C至启动的内化。内化后，细胞迅速冷却至停止内吞作用的过程和剩余的表面生物素是治疗还原剂细胞剥离。唯一保持生物素化蛋白是指那些出现在t = 0的表面和内部化，从而保护他们从剥离治疗。生物素标记的蛋白质分离出一批与链霉亲和素磁珠亲和层析和利益的蛋白质是由免疫印迹检测。 <img src="/files/ftp_upload/1669/1669fig2.jpg" alt="图2" /> 图2。 PKC的激活，增加了DAT吞率 。内在检测。 PC12细胞中稳定表达的DAT生物素，4 ° C“详细的协议”中所述。细胞被迅速加热到37 ° Ç ± 1μMPMA和孵育10&#39;，37 ° C。剥离减少残留的生物素，细胞裂解和生物素蛋白中分离链霉亲和素亲和层析柱。 （一）代表免疫印迹显示的总表面积的DAT，在t = 0（T）地带的控制权（S）和内在DAT（一）所指出的条件下。 （二）带被抓获CCD相机和量化与数量数据软件（BIO - RAD）。数据表示为％的总DAT internalized/10分钟。 <img src="/files/ftp_upload/1669/1669fig3.jpg" alt="图3" /> 图3。例免疫印迹描绘一个穷人的生物素地带 。内在检测。 PC12细胞中稳定表达的DAT生物素，4 ° C“ 详细的协议 ”中所述。细胞被迅速加热到37℃，孵育10&#39;，37 ° C。剥离减少残留的生物素，细胞裂解和生物素蛋白中分离链霉亲和素亲和层析柱。免疫印迹显示总表面的DAT在t = 0（T）地带的控制权（S）和内在DAT（一）。注意地带的控制权弄在可见光波段，表明地带效率差。

<ol>
	<li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat Neurosci. 8, 881-888 (2005).</li><li>Boudanova, E., Navaroli, D. M., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+decreases+in+dopamine+transporter+surface+expression+are+protein+kinase+C-independent.">Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent.</a> Neuropharmacology. 54, 605-612 (2008).</li><li>Boudanova, E., Navaroli, D. M., Stevens, Z., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+transporter+endocytic+determinants:+carboxy+terminal+residues+critical+for+basal+and+PKC-stimulated+internalization.">Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization.</a> Mol Cell Neurosci. 39, 211-217 (2008).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J Biol Chem. 278, 22168-22174 (2003).</li></ol>

常见问题 ：在这些实验中出现的最常见的问题是贫困地带效率。地带的效率是能够解释结果的关键。除非地带高效，是不可能得出结论，任何生物素化蛋白的内化车道，事实上，从表面上看内在​​。带≥90％的效率是最优的，和我们放弃任何结果，如果低于这个水平的地带。一个贫困地带的一个例子是如图3所示。请注意，在加沙地带车道的乐队是非常明显的，并对应一个34％的地带效率?...

measuring plasma membrane protein endocytic rates by reversible biotinylation

质膜蛋白是一种大型的，不同群体的蛋白质受体，离子通道，运输和泵组成。这些蛋白质的活性是负责各种关键的细胞活动，包括营养输送，细胞的兴奋性，和化学信号。许多质膜蛋白质吞贩运，调节通过改变蛋白的表面表达的蛋白质的功能是动态监管。促进蛋白质的内吞作用的机制，是复杂的，并不完全理解许多膜蛋白。为了充分了解的机制，控制某一特定蛋白质的内吞贩运，它是至关重要的蛋白S吞率精确测量。对于许多受体，直接吞率测量经常利用标记的受体的配体。然而，对于许多类，如运输，泵和离子通道的膜蛋白，有没有方便的配体，可以用来衡量吞率。在本报告中，我们描述了一个可逆的生物素的方法，我们聘请来衡量的多巴胺转运蛋白（DAT）的内吞率。这种方法提供了一个简单的方法来测量的内化率，并且可以很容易地雇用贩运大多数膜蛋白研究。

Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

规管的内吞作用管辖的大部分膜蛋白在细胞表面的表达水平。在这里，我们利用还原，膜impermeant生物素化试剂的多巴胺转运蛋白（DAT），一个多面体的膜蛋白的内吞率来衡量的。该方法有利于一个简单的方法来测量大多数等离子的膜蛋白的内吞率。

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

measuring-plasma-membrane-protein-endocytic-rates-reversible

Research

JoVE Journal

Biology

16.9K 次观看.  University of Massachusetts Medical School. 规管的内吞作用管辖的大部分膜蛋白在细胞表面的表达水平。在这里，我们利用还原，膜impermeant生物素化试剂的多巴胺转运蛋白（DAT），一个多面体的膜蛋白的内吞率来衡量的。该方法有利于一个简单的方法来测量大多数等离子的膜蛋白的内吞率。

视频: Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

科研

生物学

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by &Delta;&Phi;, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of &Delta;&Phi; on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of &Delta;&Phi;. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of &Delta;&Phi;.

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (&Delta;&Phi;). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the &Delta;&Phi; noninvasively. This video shows the protocol for measuring &Delta;&Phi; using di-8-ANEPPS.

诱导膜电压测量与DI - 8 - ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)1. Single-molecule videomicroscopy, offering millisecond and nanometric resolution1-11, allows a detailed representation of membrane organization12-14 by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.
We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis15,16. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.
The nanometric accuracy17 obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods17 such as STORM18, PALM19,20, RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Multiple-Target Tracing is a homemade algorithm developed for tracking individually labeled molecules within the plasma membrane of living cells. Efficiently detecting, estimating and tracing molecules over time at high-density provide a user-friendly, comprehensive tool to investigate nanoscale membrane dynamics.

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

在油脂中间相结构的测定生物大分子晶体生长的膜蛋白晶体收获和低温冷

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

膜运输过程由单一的蛋白质解析一个高度并行的纳米孔芯片分析系统

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

外周相关蛋白血浆膜分型的测定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...