Name: immunostaining-based detection of dynamic alterations in red blood cell proteins
Uploaded: 2023-03-17T13:50:00
Description: Watch this Scientific Journal Video about Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins at JoVE.com

LK感谢澳大利亚政府研究培训计划奖学金的支持。

此处描述的协议符合《赫尔辛基宣言》，并已获得德国科隆体育大学（2013 年 9 月 16 日）和格里菲斯大学（2019/808）伦理委员会的批准。对志愿者进行筛选以确保没有相关病理，并提供书面知情同意书。
1. 使用免疫组织化学方案对红细胞蛋白进行染色
注意： 材料表中提供了所需化学品和材料的详细清单。以下部分描述了所需溶液?...

<ol>
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A., Connes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide,+vasodilation+and+the+red+blood+cell.">Nitric oxide, vasodilation and the red blood cell.</a> Biorheology. 51 (2-3), 121-134 (2014).</li><li>Bor-Kucukatay, M., Wenby, R. B., Meiselman, H. J., Baskurt, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nitric+oxide+on+red+blood+cell+deformability.">Effects of nitric oxide on red blood cell deformability.</a> American Journal of Physiology. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-fractions+of+red+blood+cells+respond+differently+to+shear+exposure+following+superoxide+treatment.">Sub-fractions of red blood cells respond differently to shear exposure following superoxide treatment.</a> Biology. 10 (1), 47 (2021).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Oz&#252;yaman, B., Grau, M., Kelm, M., Merx, M. W., Kleinbongard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBC+NOS:+regulatory+mechanisms+and+therapeutic+aspects.">RBC NOS: regulatory mechanisms and therapeutic aspects.</a> Trends in Molecular Medicine. 14 (7), 314-322 (2008).</li><li>Kleinbongard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cells+express+a+functional+endothelial+nitric+oxide+synthase.">Red blood cells express a functional endothelial nitric oxide synthase.</a> Blood. 107 (7), 2943-2951 (2006).</li><li>McMahon, T. 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A., Brinkmann, C., Bloch, W., Grau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+red+blood+cell-nitric+oxide+synthase+dependent+nitric+oxide+production+during+red+blood+cell+aging+in+health+and+disease:+a+study+on+age+dependent+changes+of+rheologic+and+enzymatic+properties+in+red+blood+cells.">Increase in red blood cell-nitric oxide synthase dependent nitric oxide production during red blood cell aging in health and disease: a study on age dependent changes of rheologic and enzymatic properties in red blood cells.</a> PLoS One. 10 (4), 0125206 (2015).</li><li>Di Pietro, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+synthetic+pathway+and+cGMP+levels+are+altered+in+red+blood+cells+from+end-stage+renal+disease+patients.">Nitric oxide synthetic pathway and cGMP levels are altered in red blood cells from end-stage renal disease patients.</a> Molecular and Cellular Biochemistry. 417 (1-2), 155-167 (2016).</li><li>Grau, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Even+patients+with+mild+COVID-19+symptoms+after+SARS-CoV-2+infection+show+prolonged+altered+red+blood+cell+morphology+and+rheological+parameters.">Even patients with mild COVID-19 symptoms after SARS-CoV-2 infection show prolonged altered red blood cell morphology and rheological parameters.</a> Journal of Cellular and Molecular Medicine. 26 (10), 3022-3030 (2022).</li><li>Mozar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+nitric+oxide+synthase+modulates+red+blood+cell+deformability+in+sickle+cell+anemia.">Red blood cell nitric oxide synthase modulates red blood cell deformability in sickle cell anemia.</a> Clinical Hemorheology and Microcirculation. 64 (1), 47-53 (2016).</li><li>Ulker, P., Gunduz, F., Meiselman, H. 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M., Schindler, R., Brixius, K., Mehlhorn, U., Bloch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+circulation+activates+endothelial+nitric+oxide+synthase+in+erythrocytes.">Extracorporeal circulation activates endothelial nitric oxide synthase in erythrocytes.</a> The Annals of Thoracic Surgery. 84 (6), 2000-2003 (2007).</li><li>Horobin, J. T., Sabapathy, S., Kuck, L., Simmonds, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+stress+and+RBC-NOS+Serine1177+Phosphorylation+in+humans:+a+dose+response.">Shear stress and RBC-NOS Serine1177 Phosphorylation in humans: a dose response.</a> Life. 11 (1), 36 (2021).</li><li>Kuck, L., Grau, M., Simmonds, M. 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最近的文献高度表明，RBC-NOS蛋白对于调节RBC变形性至关重要15,22,23，这反过来又促进了它们通过狭窄的毛细血管24。蛋白质活性高度依赖于翻译后蛋白质修饰，特别是某些残基的磷酸化18。感兴趣的焦点在于磷酸化位点1177，其与RBC-NOS蛋白23的活化有关。这种蛋白质的改变已?...

红细胞 （RBC） 在心血管系统中穿行 70 至 140 天，平均红细胞年龄约为 115 天 1,2。衰老或受损的红细胞通过噬红细胞作用从循环中去除，这是一种由巨噬细胞驱动的高效清除过程3。这些细胞的预定寿命是在分化和成熟过程中放弃细胞器（包括细胞核、线粒体和核糖体）的结果之一4。因此，循环红细胞缺乏翻译机制，排除了新蛋白质的合成3。因此，对现有蛋白质的动态翻译后修饰代表了响应作用于红细胞的细胞外和细胞内应激源的急性生化调节的唯一可行机制5。
机械力似乎是导致红细胞内生化途径激活或调节的主要细胞外线索。在红细胞膜6中发现机械敏感蛋白Piezo1激发了研究这些细胞中机械激活信号传导的几条研究线7。例如，最近的进展表明，红细胞的物理性质受到蛋白质8的急性和动态变化的积极调节，其中包括翻译后磷酸化和泛素化9。由于这些正常修饰在某些疾病中有所不同9,10,11，因此确定RBC蛋白的激活状态似乎具有科学和临床意义，特别是与机械生物学过程有关。
红细胞蛋白活化状态急性变化的测定带来了一些方法学挑战。例如，储存红细胞样品以供以后分析需要保存修饰的红细胞蛋白，因为翻译后修饰是不持久的。此外，众所周知，经典的蛋白质检测方法（例如蛋白质印迹）很难在红细胞中标准化，因为相对于血红蛋白的蛋白质丰度较低，血红蛋白占这些细胞中蛋白质含量的~98%12。因此，在研究重要红细胞蛋白（例如一氧化氮合酶的红细胞特异性亚型（RBC-NOS））的急性修饰时，基于抗体的化学保存红细胞染色一直是首选方法13,14。RBC-NOS已被证明可以酶促产生一氧化氮（NO），这对于基本的RBC特性（包括RBC变形性）似乎是必不可少的15,16,17。红细胞-NOS的翻译后修饰调节催化酶活性，丝氨酸1177残基的磷酸化被描述为增加酶活性，而丝氨酸114或苏氨酸495残基的磷酸化与红细胞-NOS活性降低有关18,19。
总的来说，红细胞蛋白的临时修饰有助于重要的细胞功能，并且能够检测这些修饰蛋白的标准化方案具有很高的价值。在这里，我们提出了两种不同的方案，它们利用特异性抗体来促进RBC-NOS蛋白活化的检测，并讨论了数据分析和解释的建议。
通过测量丝氨酸1177残基处RBC-NOS磷酸化的增加来评估所描述方案的性能，以响应于反映人体脉管系统内发生的机械力（5 Pa）的机械力。

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		</tr>
		<tr>
			<td>Laser-assisted optical rotational cell analyser (LORCA)</td>
			<td>RR Mechatronics</td>
			<td></td>
			<td>Ektacytometer instrument used for shearing</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck /Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>VWR</td>
			<td>630-1985</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel(II)-sulfate Hexahydrate&#160;</td>
			<td>Sigma/Merck</td>
			<td>N4882</td>
			<td>NiSO4.6H2O</td>
		</tr>
		<tr>
			<td>Normal Goat serum</td>
			<td>Agilent/DAKO</td>
			<td>X0907</td>
			<td>NGS</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck /Millipore</td>
			<td>818715</td>
			<td>PFA</td>
		</tr>
		<tr>
			<td>Pipettes Eppendorf Reference 2</td>
			<td>VWR</td>
			<td>613-5836/ 613-5839</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-phospho eNOS Antibody (Ser1177)</td>
			<td>Merck/Millipore</td>
			<td>07-428-I</td>
			<td>Primary Antibody</td>
		</tr>
		<tr>
			<td>Reaction tubes, 2ml</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary Antibody goat anti rabbit</td>
			<td>Agilent/DAKO</td>
			<td>E0432</td>
			<td>Secondary Antibody</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Bio-Rad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>Merck /Millipore</td>
			<td>106404</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodium Dihydrogen Phosphate Monohydrate</td>
			<td>Merck /Millipore</td>
			<td>106346</td>
			<td>NaH2PO4.H2O</td>
		</tr>
		<tr>
			<td>Sodium hydroxide, 1 M</td>
			<td>Merck /Millipore</td>
			<td>150706</td>
			<td>NaOH</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)-aminomethane</td>
			<td>Merck /Millipore</td>
			<td>108382</td>
			<td>Tris</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma/Merck</td>
			<td>T7409</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20&#160;</td>
			<td>Merck /Millipore</td>
			<td>822184</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Glas microfiber filter, quality GF/F</td>
			<td>Merck /Millipore</td>
			<td>WHA1825047</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol</td>
			<td>VWR Chemicals</td>
			<td>2,89,73,465</td>
			<td></td>
		</tr>
		<tr>
			<td>&#223;-D-Glucose monohydrate</td>
			<td>Merck /Millipore</td>
			<td>14431-43-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

immunostaining-based detection of dynamic alterations in red blood cell proteins

红细胞（RBC）蛋白的抗体标记是一种常用的半定量方法，用于检测总蛋白质含量的变化或蛋白质活化状态的急性改变。它有助于评估红细胞治疗，表征某些疾病状态的差异以及描述细胞一致性。检测急性改变的蛋白质活化（例如，通过机械转导）需要足够的样品制备，以保留其他暂时的蛋白质修饰。基本原理包括固定所需红细胞蛋白的靶结合位点，以实现特异性一抗的初始结合。对样品进行进一步处理，以保证二抗与相应一抗结合的最佳条件。非荧光二抗的选择需要额外的处理，包括生物素-亲和素偶联和应用3,3-二氨基联苯胺-四盐酸盐（DAB）进行染色，需要在显微镜下实时控制染色，以便及时停止氧化，从而停止染色强度。对于染色强度检测，使用标准光学显微镜拍摄图像。在该方案的修改中，可以应用荧光素偶联的二抗，其优点是不需要进一步的开发步骤。然而，该过程需要将荧光物镜连接到显微镜上进行染色检测。鉴于这些方法的半定量性质，必须提供几种对照染色剂来解释非特异性抗体反应和背景信号。在这里，我们介绍了染色方案和相应的分析过程，以比较和讨论不同染色技术各自的结果和优势。

所提出的方案描述了促进检测红细胞蛋白急性改变的方法，并在一种众所周知的机械敏感蛋白质改变上进行了测试：丝氨酸1177残基处红细胞-NOS的磷酸化。从健康志愿者那里获得全血，随后分成两个单独的等分试样。将给定的血液样品暴露于生理量级（5 Pa）的机械剪切应力下300秒，先前显示其在丝氨酸117714处引起RBC-NOS磷酸化。在停止机械剪切暴露后，立即将血液样品固定在多聚...

Watch this Scientific Journal Video about Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins at JoVE.com

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

捕获去核红细胞蛋白质活化的动态变化带来了方法学挑战，例如保留急性刺激的动态变化以供以后评估。所提出的方案描述了样品制备和染色技术，这些技术能够保存和分析相关的蛋白质变化和随后的检测。

基于免疫染色的红细胞蛋白动态改变检测

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute ...

我们的方法通过在快速发展的机械生物学领域固定临时蛋白质修饰，然后用特异性抗体检测它们，使机械亚细胞研究成为可能。这种方法在技术上很容易应用。结果非常有效且可重复。此外，与其他技术相比，我们的方法确实可以有效地用于新兴的翻译后红细胞信号传导领域，该领域依赖于临时蛋白质修饰，尤其是红细胞机械信号传导。要进行红细胞蛋白固定，请在室温下以一比二的比例在 4% 多聚甲醛溶液中稀释全血 20 分钟。将血液溶液在室温下以132g离心三分钟，然后小心地除去上清液。将红细胞沉淀重悬于两体积的0.1摩尔PBS中，并在室温下孵育五分钟。再次，离心样品，并除去上清液，然后将红细胞沉淀重悬于一个0.1摩尔PBS体积中。用耐酒精笔标记样品ID和抗体的显微镜载玻片。加入10微升制备的红细胞溶液。将第二张载玻片以45度角放在样品上，并将样品均匀地分散在载玻片上。通过将样品悬停在本生燃烧器上并持续移动五到七秒钟来热固定载玻片。对于免疫组织化学染色，使用油脂铅笔将载玻片的 2/3 区域标记为测试，将 1/3 区域标记为对照。然后通过应用TBS清洗样品区域两次30秒。第二次洗涤后，加入0.1%胰蛋白酶，并使用带盖的专用培养室盖住载玻片。将样品在 37 摄氏度下孵育 30 分钟。在烧杯中装满自来水，并通过将烧杯中的自来水添加到载玻片上来停止酶反应。倒出溶液，用TBS清洗两个区域三次。最后一次洗涤后，加入过氧化物酶封闭溶液，并在室温下孵育30分钟。孵育后，倒出溶液并用TBS洗涤两个区域三次。然后加入3%脱脂牛奶溶液并孵育30分钟。孵育后，倒出溶液，并将一抗溶液加入测试区域。在对照区加入抗体对照溶液。将样品在四摄氏度下孵育过夜。第二天，倒出抗体溶液，并用TBS清洗两个区域三次。然后加入3%的普通山羊血清溶液以防止非特异性结合并孵育30分钟。孵育后，加入二抗溶液并孵育30分钟。倒出抗体溶液并用TBS洗涤该区域三次，然后再进行染色。接下来，要进行亲和素偶联辣根过氧化物酶反应，加入稀释的亲和素过氧化物酶溶液并孵育30分钟。将载玻片置于显微镜下，放大倍率为200倍，并将新鲜制备的DAB溶液添加到两个区域。连续监测红细胞的染色，并在背景开始着色之前用一次性移液管去除DAB溶液来停止染色。要进行样品脱水，请将载玻片放在玻璃架中，每次在增加乙醇浓度中浸入五秒钟，最后浸入二甲苯中。脱水后，将载玻片放在薄纸上，红细胞位于顶部以吸收多余的液体。在盖玻片上加入两滴或三滴安装剂，并使用盖玻片盖住载玻片。为了进行可视化和成像，请将载玻片放在与相机耦合的透射光显微镜中。打开光源和相机并启动软件。在明场模式下，通过转动粗焦和精细调焦旋钮来聚焦红细胞。同样，将红细胞聚焦在荧光模式下。要分析 RBC 灰度值，请使用 ImageJ 软件中的椭圆选择工具标记每个 RBC 的边缘。使用“测量”命令，测量来自测试的 50 个 RBC 和来自控件的 10 个 RBC 的灰度值。对于红细胞蛋白的免疫荧光染色，将样品与二级荧光偶联抗体在室温下孵育30分钟，保护其避光。孵育后，倒出抗体溶液并用TBS洗涤载玻片三次。最后一次洗涤后，将TBS留在样品上。使样品脱水并用盖玻片准备载玻片以进行显微镜评估，如图所示。打开显微镜和荧光光源，并在明场模式下聚焦红细胞。在随机选择的载玻片测试区域的至少三个不同区域中捕获明场和荧光图像。将荧光图像的曝光时间设置为一秒并捕获图像。然后切换到明场模式，将曝光时间设置为自动，并拍摄相应的明场图像。以TIFF格式保存图像。在载玻片控制区域的至少两个不同的随机选择区域中捕获荧光和明场图像。将图像保存为TIFF格式，以保留像素的原始灰度值，防止压缩，并携带采集的元数据。要测量捕获的 RBC 的灰度值，请打开 ImageJ。使用椭圆选择工具标记每个单元格，并使用“测量”命令进行分析。突出显示三到五个没有红细胞的区域，并确定灰度值以测量背景信号。要对捕获的图像执行替代数据分析，请通过斐济版本的 ImageJ 创建一个宏命令，用于给定图像的自动选择、背景校正和灰度值分析。在斐济打开 TIFF 格式图像进行分析。然后打开红细胞荧光。ijm 宏，然后单击运行。使用免疫组织化学和免疫荧光评估针对红细胞一氧化氮合酶（NOS）的抗体信号，该抗体在静止时和响应机械力暴露时在丝氨酸1177残基上磷酸化。当使用免疫组织化学或免疫荧光方法评估时，与相应的未剪切细胞相比，检测到针对丝氨酸1177残基磷酸化的RBC-NOS的抗体信号增加了三倍。用户需要确保抗体仅应用于测试区域。否则，由于缺少对照，无法分析样品。鉴于抗体种类繁多，使用这种方法可以靶向许多不同的蛋白质和信号传导过程。

Research

JoVE Journal

Biochemistry

Detection of Detergent-sensitive Interactions Between Membrane Proteins

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

科研

生物化学

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

使用碘电磷诱导红血球中的红血球

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

用细胞内快速光化学氧化蛋白质对细胞内蛋白质进行特征化

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

成熟水稻的横向切片（米 苜蓿L.）使用移液器吸头作为固定支架的扫描电子显微镜成像的内核

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

体外荧光显微镜基于动感检测的肌素特异性适应

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

活植物细胞中的Förster共振能量转移测量

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...