Name: quantitative localization of a golgi protein by imaging its center of fluorescence mass
Uploaded: 2017-08-10T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass at JoVE.com

我们要感谢 d. 斯蒂芬斯 (布里斯托尔大学, 布里斯托尔, 英国) 的 TPST1-EGFP DNA 质粒, 以及纳史木汗戈文达拉扬为帮助软件优化。这项工作得到了国家医学研究理事会 (NMRC/CBRG/007/2012)、教育部 (AcRF Tier1 rg 18/11、rg 48/13 和 RG132/15 和 AcRF Tier2 MOE2015-T2-2-073) 的资助, 以法学

注: 下面是 GLIM 的 step-by 步协议, 用于确定 EGFP 标记的 tyrosylprotein 3-o 1 (TPST1) 的 LQ, 在 HeLa 细胞中是高尔基体的驻留酶。
1. 荧光标记高尔基体微型叠的制备
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准备玻璃片
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<li>分0.3 毫升无菌 Dulbecco 改良鹰的培养基 (DMEM) 到一个24井板在一个组织培养罩的井。</li>
		<li>将Φ 12 mm no. 1.5 玻璃片用用70% 乙醇轻擦的软组织纸擦拭, 去除玻璃表面的碎片片。用一双锋?...

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A., Luini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exiting+the+Golgi+complex.">Exiting the Golgi complex.</a> Nat Rev Mol Cell Biol. 9 (4), 273-284 (2008).</li><li>Lu, L., Ladinsky, M. S., Kirchhausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisternal+organization+of+the+endoplasmic+reticulum+during+mitosis.">Cisternal organization of the endoplasmic reticulum during mitosis.</a> Mol Biol Cell. 20 (15), 3471-3480 (2009).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> J Cell Biol. 190 (2), 165-175 (2010).</li><li>Tie, H. C., 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=A+novel+imaging+method+for+quantitative+Golgi+localization+reveals+differential+intra-Golgi+trafficking+of+secretory+cargoes.">A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes.</a> Mol Biol Cell. 27 (5), 848-861 (2016).</li><li>Trucco, 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=Secretory+traffic+triggers+the+formation+of+tubular+continuities+across+Golgi+sub-compartments.">Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments.</a> Nat Cell Biol. 6 (11), 1071-1081 (2004).</li><li>Cole, N. B., Sciaky, N., Marotta, A., Song, J., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Golgi+dispersal+during+microtubule+disruption:+regeneration+of+Golgi+stacks+at+peripheral+endoplasmic+reticulum+exit+sites.">Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.</a> Mol Biol Cell. 7 (4), 631-650 (1996).</li><li>Rogalski, A. A., Bergmann, J. E., Singer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+microtubule+assembly+status+on+the+intracellular+processing+and+surface+expression+of+an+integral+protein+of+the+plasma+membrane.">Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.</a> J Cell Biol. 99 (3), 1101-1109 (1984).</li><li>Van De Moortele, S., Picart, R., Tixier-Vidal, A., Tougard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nocodazole+and+taxol+affect+subcellular+compartments+but+not+secretory+activity+of+GH3B6+prolactin+cells.">Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells.</a> Eur J Cell Biol. 60 (2), 217-227 (1993).</li><li>Nakamura, 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=Characterization+of+a+cis-Golgi+matrix+protein,+GM130.">Characterization of a cis-Golgi matrix protein, GM130.</a> J Cell Biol. 131, 1715-1726 (1995).</li><li>Roth, J., Berger, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+localization+of+galactosyltransferase+in+HeLa+cells:+codistribution+with+thiamine+pyrophosphatase+in+trans-Golgi+cisternae.">Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.</a> J Cell Biol. 93 (1), 223-229 (1982).</li><li>Baeuerle, P. A., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+sulfation+is+a+trans-Golgi-specific+protein+modification.">Tyrosine sulfation is a trans-Golgi-specific protein modification.</a> J Cell Biol. 105 (6), 2655-2664 (1987).</li><li>Honda, A., Al-Awar, O. S., Hay, J. C., Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+Arf-1+to+the+early+Golgi+by+membrin,+an+ER-Golgi+SNARE.">Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE.</a> J Cell Biol. 168 (7), 1039-1051 (2005).</li><li>Lavieu, G., Zheng, H., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stapled+Golgi+cisternae+remain+in+place+as+cargo+passes+through+the+stack.">Stapled Golgi cisternae remain in place as cargo passes through the stack.</a> Elife. 2, 00558 (2013).</li><li>Zhao, X., Lasell, T. K., Melancon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+large+ADP-ribosylation+factor-guanine+nucleotide+exchange+factors+to+different+Golgi+compartments:+evidence+for+distinct+functions+in+protein+traffic.">Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic.</a> Mol Biol Cell. 13 (1), 119-133 (2002).</li><li>Dejgaard, S. Y., Murshid, A., Dee, K. M., Presley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy-based+linescan+methodologies+for+intra-Golgi+localization+of+proteins.">Confocal microscopy-based linescan methodologies for intra-Golgi localization of proteins.</a> J Histochem Cytochem. 55 (7), 709-719 (2007).</li><li>Fourriere, L., Divoux, S., Roceri, M., Perez, F., Boncompain, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-independent+secretion+requires+functional+maturation+of+Golgi+elements.">Microtubule-independent secretion requires functional maturation of Golgi elements.</a> J Cell Sci. 129 (17), 3238-3250 (2016).</li><li>Volchuk, 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=Countercurrent+distribution+of+two+distinct+SNARE+complexes+mediating+transport+within+the+Golgi+stack.">Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack.</a> Mol Biol Cell. 15 (4), 1506-1518 (2004).</li><li>Popoff, V., Adolf, F., Brugger, B., Wieland, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+budding+within+the+Golgi+stack.">COPI budding within the Golgi stack.</a> Cold Spring Harb Perspect Biol. 3 (11), 005231 (2011).</li></ol>

以前, 在光显微镜下的高尔基体蛋白的定位, 主要是通过与已知的高尔基体标记的图像相关或重叠程度的蛋白质的图像进行量化,15,16, 17。由此产生的相关或重叠系数反映了测试蛋白在空间上对高尔基标记的接近程度。此方法至少有三警告。首先, 相关或重叠系数是非线性的, 不直接表示空间距离。第二, 相关性的程度严重依赖于?...

高尔基复合体在哺乳动物细胞分泌/吞的蛋白质和脂质 (以下) 的贩运中起着重要作用1,2,3。在高尔基, 货物不仅排序到各种细胞舱室, 但也修改了不同类型的糖基化。哺乳动物高尔基复合体包括许多侧向连接的高尔基栈, 通常由 4-11 紧密相邻和扁平膜囊称为泡。连续堆叠高尔基体泡进一步分类, 从一端到另一个, 作为cis, 内侧和反式-泡。在高尔基体的反式一侧,反式-大多数膜囊发展成管状和网状膜网络, 称为反式-高尔基网络 (TGN)4。在分泌通路中, 来自内质网 (ER) 的货物在其cis端进入高尔基体, 然后依次通过内侧和反式-泡。货物最终退出高尔基体在反式-高尔基或 TGN 注定的细胞膜, 体或分泌颗粒。
的分子和细胞机制如何运输的高尔基体和高尔基体保持其池组织仍然神秘, 目前仍在激烈辩论1。这一领域的难点之一是, 高尔基泡只能在电子显微镜下解决 (EM), 因为光学显微镜 (200 nm) 的分辨率不足以解决单个高尔基泡 (< 100 纳米池厚度和距离)。因此, 亚高尔基定位的居民蛋白和转运货物是常规确定的免疫金 EM。然而, 免疫金 EM 是非常严格的技术要求, 它是超出了能力的大多数细胞生物学实验室。虽然 em 的分辨率可以 sub-nanometer, 免疫金 em 所提供的分辨率很大程度上受阻于抗体复合体 (主辅抗体) 和金粒子的大小, 而且可能比 20 nm 差。此外, EM 图像是从2D 薄剖面, 而不是一个3D 的全球视野的高尔基, 这可能导致错误的结论取决于相对位置和方向的2D 节5。例如, 研究 EM 开路不能可靠地区分一个小泡从一个小管的正交视图, 因为两者都可以显示相同的圆形膜剖面。最近出现的超分辨率显微镜技术, 如 3 d 结构照明显微镜 (3 d SIM), 刺激发射损耗 (STED), 光定位显微镜 (手掌) 和随机光学重建显微镜 (风暴), 使得有可能解决亚高尔基体在光显微镜下的结构6。然而, 至少有四缺点, 可以极大地限制他们的使用在细胞生物学研究高尔基。1) 目前超分辨率技术需要昂贵和特殊的硬件配置, 这是超出大多数细胞生物学实验室。2) 某些超分辨率技术需要特殊的荧光标记协议。3) 虽然在最佳条件下, 这些技术在空间分辨率上要求 20-110 nm, 但实际样品中获得的实用分辨率可能更差。4) 与常规显微镜相比, 这些超分辨率技术在进行多色、3D 或活细胞成像方面仍有困难, 无论是单独还是联合进行。可能最重要的是, 免疫金 EM 和超分辨显微镜技术的产生定性而不是定量的本地化数据。
试图部分解决上述问题, 我们最近开发了一个传统的光学显微镜的方法, 这是命名为高尔基体蛋白质的成像中心的质量 (GLIM), 系统和定量本地化高尔基蛋白质的分辨率相当于免疫金 EM7。在这种方法中, 高尔基体在培养的哺乳动物细胞被分散作为高尔基体 mini-stacks 通过治疗 nocodazole, 一个微管聚药物。广泛的研究表明, nocodazole 诱导高尔基 mini-stacks (以下高尔基体 mini-stacks) 在组织和细胞功能上都与本机的基尔基体紧密类似8,9,10, 11。测试蛋白的定位商 (LQ) 可以通过 GLIM 获得, 它表示定量的亚尔基体定位。对 LQs 的数值进行了比较, 得到了超过25高尔基标记的 LQ 数据库。
在 GLIM, 高尔基 mini-stacks 是三重标记的内生或外部表达 GM130, 高尔特 mCherry 和测试蛋白 (x)。GM130 和高尔特-mCherry, cis-和反式-高尔基标记分别12,13, 提供参考点。三重荧光, 红色 (R), 绿色 (G) 和远红色 (B), 被人为地显示红色, 绿色和蓝色, 分别。采用荧光质量中心 (以下简称中心) 实现亚像素分辨率。高尔基轴被定义为从 GM130 中心到高尔 mCherry 的向量。高尔基 mini-stack 被建模为一个圆柱形结构, 在高尔基轴周围有无限的旋转对称。因此, 高尔基体 mini-stack 可以被进一步建模为沿高尔基轴的 one-dimensional 结构。测试蛋白 x 的 LQ 定义为 dx/d1, 其中 dx是从 x 中心到 GM130 的距离, 而 d1是从 mCherry 中心到 GM130 的距离。如果 x 轴的中心是轴向的, 它的投影轴线距离用于计算。变量, 包括高尔基轴, 轴向角, dx, d1, 角α和角β, 为 GLIM 的示意图在图 1中说明。LQ 是独立的高尔基轴向角, 虽然高尔基 mini-stacks 东方随机在一个细胞。
高尔基体 mini-stacks 在图像中呈现不均匀。我们制定了三标准, 以选择分析高尔基 mini-stacks GLIM。1) 信噪比判据, 在每个通道中, 高尔基体 mini-stack 的总强度与背景的标准差 (SD) 的比值为≥30。这一标准是确保质量中心的定位精度, 这取决于高尔基体 mini-stacks 的信噪比。2) 轴向角度或距离标准, 要求 d1≥ 70 nm。d1随着高尔基轴向角的增大而减小。当轴向角度接近90°或垂直方向时, mini-stack 变为 non-resolvable, 因为 d1接近0。d1≥ 70 nm 可以有效地排除垂直高尔基体附近的 mini-stacks。3) co-linearity 标准, 其中任一 | tan α |或 | tan β |≤0.3。这一标准确保了 mini-stack 的三中心足以共我们的 one-dimensional 模型的高尔基 mini-stack。所有的光显微镜都有色差, 严重地扭曲了红色、绿色和远红光荧光中心的相对位置。对显微镜系统的色差进行了实验标定, 成像110纳米珠, 这是红, 绿和远红色荧光三标。对于每个珠子图像, 红色中心被定义为珠的真实位置, 绿色和远红色中心的色移由一阶多项式函数拟合。高尔基体 mini-stacks 的中心受多项式函数的作用, 以纠正绿色和远红通道中的色移。
通过 GLIM, 我们可以在标准条件下在高尔基轴上实现约 30 nm 的分辨率。重要的是, 它提供了一种系统的方法来定量绘制任何高尔基蛋白。GLIM 可由常规显微镜 (如广域或共聚焦显微镜) 使用普通荧光标记协议进行。图像处理和数据处理可能会缩短一小时。通过 GLIM, 我们直接演示了分泌货物从cis-到高尔基体7的跨端的逐步过渡。

<table>
	<tbody>
		<tr>
			<td class="xl67">fluorescence beads. Commercial name: TetraSpeck beads</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">T7279</td>
			<td class="xl67">As multi-color beads to calibrate chromatic-shift of the microscope.</td>
		</tr>
		<tr>
			<td class="xl67">Glass coverslip &#934; 12 mm (No. 1.5)</td>
			<td class="xl67">Menzel</td>
			<td class="xl75">CB00120RAC</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Glass coverslip &#934; 25 mm (No. 1.5)</td>
			<td class="xl67">Menzel</td>
			<td class="xl75"></td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">DMEM</td>
			<td class="xl67">Capricon</td>
			<td class="xl75">DMEM-HPA-P50</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Trypsin-EDTA</td>
			<td class="xl67"></td>
			<td class="xl75"></td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">FBS</td>
			<td class="xl67">GE Hyclone</td>
			<td class="xl75">SV30160.03</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Nocodazole</td>
			<td class="xl67">Merck</td>
			<td class="xl75">487928</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">transfection reagent. Commercial name: Lipofectamine 2000</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">11668-019</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">transfection medium. Commercial name: OptiMEM</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">31985070</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">TPST1-EGFP</td>
			<td class="xl67">Addgene</td>
			<td class="xl75">66617</td>
			<td class="xl67">A gift from D. Stephens (University of Bristol, Bristol, United Kingdom)</td>
		</tr>
		<tr>
			<td class="xl67">GalT-mCherry</td>
			<td class="xl67"></td>
			<td class="xl75"></td>
			<td class="xl67">Made in our lab.</td>
		</tr>
		<tr>
			<td class="xl67">paraformaldehyde</td>
			<td class="xl67">Merck</td>
			<td class="xl75">1.04005.1000</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">saponin</td>
			<td class="xl67">Sigma-Aldrich</td>
			<td class="xl75">47036</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">poly(vinyl alcohol) (Mw ~31,000). Commercial name: Mowiol-488</td>
			<td class="xl67">CALBIOCHEM</td>
			<td class="xl75">475904</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">BSA</td>
			<td class="xl67">Sigma-Aldrich</td>
			<td class="xl75">A9647</td>
			<td class="xl67"></td>
		</tr>
		<tr>
			<td class="xl67">Mouse anti-GM130</td>
			<td class="xl67">BD Biosciences</td>
			<td class="xl75">610823</td>
			<td class="xl67">Primary antibody for human GM130</td>
		</tr>
		<tr>
			<td class="xl67">far-red fluorescence conjugated goat anti-mouse IgG. Commercial name: Alexa Fluor 647 conjugated goat anti-mouse IgG</td>
			<td class="xl67">Invitrogen</td>
			<td class="xl75">A-21235</td>
			<td class="xl67">Far red fluorescence conjugated secondary antibody</td>
		</tr>
	</tbody>
</table>

quantitative localization of a golgi protein by imaging its center of fluorescence mass

高尔基复合体由连续堆积的膜泡组成, 可进一步划分为亚高尔基区, 包括cis-高尔基体、中高尔基、反式-高尔基和反式-高尔基网络。高尔基体的细胞功能由其驻留蛋白的特征分布决定。传统光学显微镜的空间分辨率过低, 无法解决亚高尔基结构或泡。因此, 免疫金电子显微镜是一种选择的方法, 本地化的蛋白质在亚高尔基水平。然而, 该技术和仪器超出了大多数细胞生物学实验室的能力。我们在这里描述我们最近开发的超分辨率的方法称为高尔基蛋白的成像中心的质量 (GLIM) 系统和定量本地化高尔基体蛋白。GLIM 是基于标准荧光标记协议和传统的广域或共聚焦显微镜。它涉及对显微系统的色移像差的标定、图像采集和后分析。将测试蛋白的亚尔基体定位定量地表达为定位商。GLIM 有四主要优点;它是快速的, 根据传统的方法和工具, 本地化的结果是定量的, 它提供了〜 30 nm 的实际分辨率沿高尔基轴。在这里, 我们描述了 GLIM 的详细协议, 本地化一个测试高尔基蛋白。

现代研究级光学显微镜配有计划物镜透镜, 如我们实验室使用的, 显示最小色差 (图 2a)。然而, 仔细检查多色荧光珠图像可以揭示同一珠子的不同颜色图像的移位 (图 2B)。我们定义红色通道没有色差, 因此红色荧光的中心是珠的真实位置。绿色和远红光荧光的相对色位移可以用来自红色荧光中心的绿色和?...

Watch this Scientific Journal Video about Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass at JoVE.com

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

高尔基体的精确定位对于了解高尔基体的细胞功能是至关重要的。然而, 传统的光学显微镜无法解决亚尔基体的结构。在这里, 我们描述了一个传统的基于显微镜的超分辨率方法的协议, 定量地确定蛋白质的亚高尔基体定位。

高尔基蛋白质荧光质量中心的定量定位

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

Research

JoVE Journal

Biology

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

采用先进的3D荧光显微镜定量分析自噬

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

快速分析和荧光显微镜图像的探索

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...