Name: pip-on-a-chip: a label-free study of protein-phosphoinositide interactions
Uploaded: 2017-07-27T13:00:00
Description: Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

部分支持DS和CEC的授权AI053531（NIAID，NIH）; SS和PSC由授权N00014-14-1-0792（ONR）支持。

1.清洁玻璃盖板<ol><li>稀释7倍的清洁溶液（参见材料表 ）用100毫米深硼硅酸盐玻璃皿的去离子水将其稀释7倍，并将其在平板热板上加热至95℃，持续20分钟，或直到混浊溶液变澄清。 注意：解决方案将会很热，请注意避免身体受伤。 7x清洁溶液是[氧化 - （氧化（膦酰氧基）磷酰基]氧磷酰基]磷酸六钠，2-（2-丁氧基乙氧基）乙醇，1,4-双（2-乙基己氧基）-1,4-二氧代丁烷钠-2-磺酸?...

每个PIP变体虽然浓度低，但存在于特定细胞器的细胞溶质表面，其中它们有助于建立独特的物理组成和细胞器膜1的功能特异性。一个的PIP的最重要的用途是作为一个特定的对接平台需要特定亚细胞定位和/或活化6,7蛋白质的众人。由于其在细胞生理学和疾病中的作用，在生理学相关的背景下研究体外蛋白质-PIP相互作用的能力是?...

无数的相互作用和生物化学过程发生在二维流体的膜表面上。真核细胞中的膜封闭的细胞器不仅在生物化学过程及其相关的蛋白质组学中而且在其脂质组成中是独特的。一类特殊的磷脂是磷酸肌醇（PIP）。即使它们包括只有1％的细胞脂质组的，它们在信号转导，自噬和膜运输至关重要的作用，其中包括1，2，3，4。通过细胞PIP激酶肌醇头部基团的磷酸化动态产生七个PIP头基是单- ，双- ，或三-磷酸化5。另外，PIP定义膜的亚细胞同一性，并且用作含有一种或多种磷酸苷酶的蛋白质/酶的专门的膜对接位点itide结合结构域，例如，pleckstrin同源（PH），PHOX同源性（PX），和底物蛋白N端同源性（ENTH）6,7。最好研究的PIP​​结合结构域之一是在高纳摩尔 - 低微摩尔范围亲和力8内与磷脂酰肌醇4,5-二磷酸（PI（4,5）P 2 ）特异性相互作用的磷脂酶C（PLC）-δ1PH结构域，9，10，11。 已经开发了多种定性和定量的体外方法，用于研究这些相互作用的机制，热力学和特异性。在最常用的PIP结合测定中，表面等离子体共振（SPR），等温量热法（ITC），核磁共振（NMR）光谱，脂质体浮选/沉降测定和脂质印迹（脂肪印迹/ PIP条）12，13。即使这些被广泛使用，它们都具有许多缺点。例如，SPR，ITC，和NMR需要大量的样品，昂贵的仪器，和/或受过训练的人员12，13的。一些测定法形式，例如基于抗体的脂质的印迹利用的PIP的水可溶形式，并在非生理方式12，14，15，16呈现它们。此外，脂质印迹不能可靠定量，它们经常导致假阳性/阴性的观察12，17，18。为了克服这些挑战并改进当前的工具集，在上下文中基于支持的脂质双层（SLB）建立了一种新的无标记方法<html> icrofluidic平台，将其成功地应用于蛋白质-PIP相互作用的研究（ 图1）19。 用于检测蛋白质-PIP相互作用的策略是基于pH调节感测。这涉及一种pH敏感的染料，其具有直接与磷脂酰乙醇胺脂质头部组20缀合的邻 -磺酰罗丹明B（ o SRB）。 o SRB-POPE探针（ 图2A ）在低pH下是高度荧光的，在高pH下淬灭，在7.5mol％含PI（4,5）P 2的SLB中具有约6.7的pKa（ 图5B ）。 PLC-δ1PH结构域已被广泛使用，用于验证蛋白质-PIP结合方法由于其对PI（4,5）P 2（ 图5A）高特异性21,22“&gt; 23，24，25 .Hence，我们的理由是，PLC-δ1PH结构域可以用来测试其结合至PI（4,5）P 2通过PIP-上的单芯片测定法。该PH结构域构建体在此研究中使用具有净正电荷（等电点8.4），并且因此吸引OH -离子（ 图5C）在结合PI（4,5）P 2含SLBs中，PH结构域所带来的OH -离子的膜表面，这反过来又调制所述界面电势并转移öSRB-POPE（ 图5C）26，随着PH结构域浓度的函数，荧光被猝灭（ 图6A）的质子化状态。最后，将归一化的数据是适合于结合等温线以确定PH结构域-P1（4,5）P 2相互作用的亲和力（ 图6B ， 6C ）。/ P&gt; 在这项研究中，提供了详细的方案，以在微流体平台内进行蛋白质结合含PIP的SLBs。该协议使读者将微流体装置和囊泡制备装配成SLB形成和蛋白质结合。此外，提供了用于提取PLC-δ1PH域-PI（4,5）P 2相互作用的亲和度信息的数据分析方向。

<table border="0" cellpadding="0" cellspacing="0" width="1638">
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		<tr height="20">
			<td height="20" style="height:20px;width:363px;">Coverslip</td>
			<td style="width:156px;"></td>
			<td style="width:123px;"></td>
			<td style="width:997px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Glass Coverslips: Rectangles</td>
			<td>Fisher Scientific</td>
			<td>12-544B</td>
			<td>22 x 40 x 0.16 - 0.19 mm, No. 1 1/2; Borosilicate Glass</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7X Cleaning Solution</td>
			<td>MP Biomedicals</td>
			<td>976670</td>
			<td>Detergent</td>
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		<tr height="20">
			<td height="20" style="height:20px;">PYREX Crystallizing Dish</td>
			<td>Corning</td>
			<td>3140-190</td>
			<td>Borosilicate glass dish with a flat bottom; Diameter x Height (190 x 100 mm); Distributor: VWR (89090-700)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sentry Xpress 2.0</td>
			<td>Paragon Industries</td>
			<td>SC-2</td>
			<td>Kiln</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
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			<td>Catalog Number</td>
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			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning&#160;</td>
			<td>4019862</td>
			<td>Polydimethylsiloxane (PDMS); Distributor: Ellsworth Adhesives</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PYREX Desiccator</td>
			<td>VWR</td>
			<td>89134-402</td>
			<td>Vacuum Rated</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Biopsy punch</td>
			<td>Harris</td>
			<td>15110-10</td>
			<td style="width:997px;">Harris Uni-Core; 1.0 mm diameter; Miltex Biopsy Punch with Plunger (Cat. No. 15110-10) can be used as an alternative</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Plasma Cleaning System</td>
			<td>PlasmaEtch</td>
			<td>PE25-JW</td>
			<td>2-stage Direct Drive Oil Vacuum Pump, O2 service (Krytox Charged)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital Hot Plate</td>
			<td>Benchmark</td>
			<td>H3760-H</td>
			<td>Purchased through Denville Scientific (Cat. No. 1005640)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Frosted Micro Slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Frosted, Selected, and Precleaned; Made of Swiss Glass; Thickness: 1 mm; Dimensions: 75 x 25 mm; GR 144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
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			<td height="20" style="height:20px;">Mold</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">AutoCAD</td>
			<td>Autodesk</td>
			<td>v.2016</td>
			<td>Drafting software for the photomask design</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomask</td>
			<td>CAD/Art Services</td>
			<td>N/A</td>
			<td>Design with black background and clear features was printed at 20k dpi resolution on a transparent mask (5 x 7 in) by CAD/Art Services</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicone Wafers</td>
			<td>University Wafer</td>
			<td>1575</td>
			<td>Prime Grade, Single Side Polished; 100 mm (4 inch) Diameter; 525 um Thickness</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 50</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Negative Tone Photoresist; Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU-8 Developer</td>
			<td>MicroChem Corp.</td>
			<td>N/A</td>
			<td>Penn State Nanofabrication Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="20">
			<td height="20" style="height:20px;">SUV</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>850457C</td>
			<td>POPC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4-phosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>840045X</td>
			<td>PI4P</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-&#945;-phosphatidylinositol-4,5-bisphosphate&#160;</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X</td>
			<td>PI(4,5)P2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine</td>
			<td>Avanti Polar Lipids</td>
			<td>850757C</td>
			<td>POPE; Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lissamine Rhodamine B Sulfonyl Chloride (mixed isomers)</td>
			<td>ThermoFisher Scientific</td>
			<td>L-20</td>
			<td>Required for the synthesis of oSRB-POPE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pH Sensitive Fluorescent Lipid Probe (oSRB-POPE)</td>
			<td>In-house</td>
			<td>N/A</td>
			<td>In-house Synthesis (Huang D. et al. 2013)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Scintillation Vial</td>
			<td>VWR</td>
			<td>66022-065</td>
			<td>20 mL volume capacity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aquasonic 250D</td>
			<td>VWR</td>
			<td>N/A</td>
			<td>Ultrasonic Water Bath</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclepore Track-Etched Membranes</td>
			<td>Whatman</td>
			<td>110605</td>
			<td>Polycarbonate Membrane; Diameter: 25 mm; Pore Size: 0.1 um; Distributor: Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>VWR</td>
			<td>CX1054-6</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LIPEX Extruder</td>
			<td>Transferra Nanosciences</td>
			<td>T.001</td>
			<td>LIPEX 10 mL Thermobarrel Extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viscotek 802 DLS</td>
			<td>Malvern Instruments</td>
			<td>N/A</td>
			<td>Dynamic Light Scattering; Penn State X-Ray Crystallography Facility Property</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>v.6</td>
			<td>Curve-fitting software for data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axiovert 200M Epifluorescence Microscope</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioCam MRm Camera</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Camera</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">X-Cite 120</td>
			<td>Excelitas Technologies</td>
			<td>N/A</td>
			<td>Light Source</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa 568 Filter Set</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Ex/Em 576/603 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AxioVision LE64 v.4.9.1.0 Software</td>
			<td>Carl Zeiss Microscopy</td>
			<td>N/A</td>
			<td>Image Processing Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>10034-132</td>
			<td>200 uL pipette tips; Thin and smooth tip for applying the protein solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tips</td>
			<td>VWR</td>
			<td>53509-070</td>
			<td>10 uL pipette tips; Thin and smooth tip for applying the vesicle solution into the microfluidic channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion Star A321 pH meter</td>
			<td>Thermo Scientific</td>
			<td>STARA3210</td>
			<td>pH meter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Orion micro pH probe</td>
			<td>Thermo Scientific</td>
			<td>8220BNWP</td>
			<td>micro pH probe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-(2-Hydroxyethyl)-Piperazine-N&#39;-(2-Ethanesulfonic Acid)</td>
			<td>VWR</td>
			<td>VWRB30487</td>
			<td>HEPES, Free Acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride</td>
			<td>VWR</td>
			<td>BDH8014-2.5KGR</td>
			<td>NaCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Allied Wire &#38; Cable</td>
			<td>TFT-200-24 N</td>
			<td>Internal Diameter: 0.020-0.026 inches (0.051-0.066 cm); Wall Thickness: 0.010 inches (0.025 cm); Flexible Polytetrafluoroethylene Thin-Wall Tubing; Natural Color</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nitrogen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxygen Gas - Industrial</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquid Nitrogen</td>
			<td>Praxair</td>
			<td>N/A</td>
			<td>Local Provider</td>
		</tr>
	</tbody>
</table>

pip-on-a-chip: a label-free study of protein-phosphoinositide interactions

许多细胞蛋白质与膜表面相互作用以影响基本的细胞过程。这些相互作用可以针对膜内的特定脂质组分，如在磷酸肌醇（PIP）的情况下），以确保特定的亚细胞定位和/或活化。已广泛研究PIP和细胞PIP结合结构域以更好地了解其在细胞生理学中的作用。我们对支持的脂质双层（SLB）进行了pH调节测定，作为研究蛋白质-PIP相互作用的工具。在这些研究中，pH敏感的邻 -磺酰罗丹明B缀合的磷脂酰乙醇胺用于检测蛋白质-PIP相互作用。当蛋白质与含PIP的膜表面结合时，调节界面电位（ 即局部pH值的变化），使探针的质子化状态发生变化。通过使用磷脂酶C delta1 Pleckstr提出了pH调节测定的成功应用的案例研究在同源性（PLC-δ1PH）结构域和磷脂酰肌醇4,5-二磷酸（PI（4,5）P 2 ）相互作用为例。这种相互作用的表观解离常数（ K d，app ）为0.39±0.05μM，类似于其他人获得的K d值。如前所述，PLC-δ1PH结构域是PI（4,5）P 2特异性，显示与磷脂酰肌醇4-磷酸的结合较弱，并且不与纯磷脂酰胆碱SLB结合。 PIP片上测定优于传统的PIP结合测定，包括但不限于低样品体积和无配体/受体标记要求，测试高和低亲和力膜相互作用的能力与小和大分子，提高信噪比。因此，PIP-on-a-chip方法的使用将有助于阐明各种膜相互作用的机制。此外，这种方法可能是你的确定调节蛋白质与膜相互作用的能力的治疗剂。

我们使用pH调节测定法研究了PIP-on-a-chip微器件中的PLC-δ1PH域-PI（4,5）P 2相互作用（ 图1 ）。通过详细的方案，我们演示了如何准备和组装微流体装置组件，制备小单层囊泡（SUV）（ 图2 ），在装置内形成SLB（ 图3 ），并测试蛋白质与含有PIP的SLB的结合。 图4中描绘了典型的SLB...

Watch this Scientific Journal Video about PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions at JoVE.com

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

我们在微流体平台的背景下提供支持的脂质双层，以使用基于pH调节的无标记方法研究蛋白质 - 磷酸肌醇相互作用。

PIP-on-a-chip：蛋白质 - 磷酸肌醇相互作用的无标记研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

The overall goal of the PIP-on-a-chip assay is to assess protein membrane interactions in a quantitative label-free manner. Protein membrane interactions are the heart of so many processes of the cell and its pathogens, but techniques to study these interactions are few. The advantages of this technique are low simple volume, no ligand or receptor labeling requirements combined with the ability to test membrane interactions in a physiologically relevant manner.Many therapeutic targets of viruses are membrane proteins, studies of these target proteins are often performed in solution using detergents. Our technique provides a more biologically relevant alternative. While you will see that this assay is capable of monitoring the interactions of proteins with membranes, it's actually quite a versatile assay and can be used to monitor iron membrane, small molecule membrane and even peptide membrane interactions.To begin, mix polydimethylsiloxane or PDMS prepolymer and the curing agent at a 10:1 ratio, in a large plastic weigh boat. Degas the mixture in a vacuum for one hour with the vacuum strength at 500 tor or less. Place the silicon master which contains multiple replicates of the same SU8 micropattern in a large plastic weigh boat and pour in the degas PDMS, then, cure it in a dry oven at 60 degrees celsius overnight.The next day, gently peel off the PDMS from the silicon master using one's hands. Mark the boundaries of each micro pattern in rectangles using a surgical scalpel and a ruler. Then, cut the PDMS into blocks, punch 16 holes at both ends of each micro channel with a biopsy punch, to make holes with 1.0 millimeter diameter.Pipette calculated volumes of phosphatidylcholine, Phosphatidylinositol 4, 5-bisphosphate and pH sensitive fluorescent probe into a single 20 milliliter glass ventilation vial. Dry the mixture in a stream of nitrogen gas inside the chemical fume hood for 10 minutes or until the solvent evaporates and the thin lipid film forms at the bottom of the vial. Then, desiccate the mixture under vacuum for at least three hours at a vacuum strength of 10 millitor to remove any residual organic solvent.Rehydrate the dried lipid film with five milliliters of running buffer, place the rehydrated lipid in an ultrasonic bath at an operating frequency of 35 kilohertz for 30 minutes at room temperature. Freeze thaw the vesicle suspension with liquid nitrogen and the 40 degree celsius water bath to get unilaminar vesicles. Repeat the freeze thaw 10 times, extrude the vesicle suspension to a 0.1 micron track edge polycarbonate membrane, using a lipid extruder to enrich for small unilaminar vesicles.Repeat the extrusion 10 times. Test the inlets and outlets of the PDMS block for blockage by squirting de-ionized water through the holes using a water wash bottle, then, dry the PDMS block with nitrogen gas. Next, place the PDMS block and the pre-cleaned cover slip inside the oxygen plasma system sample chamber.Expose the PDMS block and the cover slip with oxygen plasma for 45 seconds with the power settings at 75 watts, oxygen flow speed at 10 cubic centimeters per minute and the vacuum strength of 200 millitor. Next, place the pattern surface of the PDMS block in the contact with the cover slip immediately after the oxygen plasma treatment. Press gently to remove any air bubbles at contact sites.Place the device on a level hot plate at 100 degree celsius for three minutes to enhance the bonding. Use a wet lint-free wipe with 100%ethanol to remove any dust particles from the top and the bottom of the device. Then, tape the device on top of a glass microscope slide.Transfer 100 microliters of the Phosphatidylinositol 4, 5-bisphosphate containing small unilaminar vesicles into a 0.65 milliliter microcentrifuge tube. Adjust the pH of the solution to approximately 3/2 by adding 6.4 microliters of 0.2 normal hydrochloric acid. Pipette 10 microliters of the pH adjusted small unilaminar vesicle solution into each channels through the inlet and apply pressure through the pipette until the solution reaches the outlet.Detach the tip from the pipette and leave it attached to the device. After repeating this step for each channel, incubate the device for 10 minutes at room temperature, injection of vesicles into micro channels should be performed immediately after the device assembly. Meanwhile, cut sets of inlet and outlet tubing, using tweezers, connect the outlet tubing set to the device and then tape the device onto a microscope stage.Submerge one end of the inlet tubing set in 25 milliliters of running buffer contained in a conical tube and tape it to make sure that the tubing is secured. Using a lab jack, place the conical tube on a higher ground than the device in order to push the solution through the micro channels via gravity flow. For each inlet tube, use a syringe to draw one milliliter of running buffer from the free end of the tubing.Remove the pipette tip from the inlet and insert the free end of the inlet tubing into the device. Repeat this process to connect all the inlet tubing pieces to the device. Flowing running buffer through the channels helps to remove excess unruptured vesicles and equilibrate the bilayer to experimental conditions.Next, open the microscope control software. On the left panel, click on the microscope tab and choose the 10x objective. Click on live and then the Alexa 568 image icons on the toolbar, using the fine and coarse adjustment knobs, focus on the micro channels.Scan through the device to check through the quality of the SLBs and the channels. Then, click the FL shutter closed image icon on the toolbar, click on the acquisition tab and under basic adjustments, select exposure time. Set the exposure time to 200 milliseconds.On the left panel, click on multi-dimensional acquisition. Under the filters menu, select the red channel. Then, click on the timelapse menu, set the time interval to five minutes, duration to 30 minutes and lapse menu.Select the circle tool under the measure tab and draw a circle in any channel. Right click while the circle is selected and choose properties. Under the profile tab, check all T to view the fluorescence intensity as a function of time.Make sure this curve reaches a plateau which indicates equilibrium before proceeding to the next step, lower the buffer solution to an equal ground as the device, to stop the flow. One at a time, detach each outlet tubing and apply 200 microliters of each protein dilution into the outlet channel using a pipette. Do not apply any pressure, let gravity do the work.Detach the tip from the pipette and leave it attached to the micro fluidic device. Repeat this process for each channel and make sure that air bubbles are not introduced into the channels during this process. Next, lower the inlet tubing to a ground below the microfluidic device to start flowing the protein through the micro channels.Tape the free end of the tubing to a waste container. Flow the dilutions of the pleckstrin homology domain for 30 minutes. On the left panel of the software, under the timelapse tab, click on start, to begin imaging again.Shown here, is a representative view of the Phosphatidylinositol 4, 5-bisphosphate containing SLBs within micro channels. Before, and after, adding the pleckstrin homology domain at the indicated concentrations. Fluorescence intensities from the line scanned across micro channels are plotted as a function of distance and pixels for phosphatidylcholine, Phosphatidylinositol 4 phosphate, and Phosphatidylinositol 4, 5-bisphosphate binding experiments.Then, normalize binding data from individual experiments, is plotted as a function of phospholipase C delta 1 pleckstrin homology domain concentration and fit to a binding isoterm to extract apparent association constants. Comparison of the apparent association constants, shows that phospholipase C Delta 1 pleckstrin homology domain interacts with Phosphatidylinositol 4, 5-bisphosphate specifically, as expected. After watching this video, you should have a good understanding of how to make small unilaminar vesicles, make microfluidic devices, form supported lipid bilayers inside these microfluidic devices, as your protein membrane binding interactions, using this assay with our PIP-on-a-chip approach.Once mastered, this technique can be done in three hours if it is performed properly. Following this procedure, other methods like fluorescence recovery after photo bleaching can be performed in order to assess the effect of protein membrane binding on the lateral diffusion of the lipids. This technique paves the way to study protein membrane interactions with the assemble of lipids found in cells instead of one at a time, this approach will therefore broadly impact biochemistry and cell biology of protein membrane interactions.

Research

JoVE Journal

Bioengineering

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Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

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科研

生物工程

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内皮化微流体研究在血液病的微血管相互作用

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非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

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使用无标记光学生物传感器由G-蛋白偶联受体，以检测钾通道的调制

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微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

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无标记单分子检测使用微型环光学谐振器

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

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3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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用无标签荧光显微镜评价活体、人体阻力动脉中胶原蛋白和弹性蛋白的压力依赖性 Microarchitectures

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