定量免疫检测，以衡量在中心体的变异蛋白水平
Summary
Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein’s fluorescence intensity to that of an appropriate internal standard.
Abstract
中心体是充当有丝分裂纺锤体的两极保持基因组的完整性，或装配初级纤毛以促进感觉功能在细胞中的小但很重要的细胞器。一个蛋白的水平可在中心体比其他.cellular位置来调节不同的，并且在一些蛋白在不同点的细胞周期的中心体水平的变化似乎是中心粒装配的适当调节是至关重要的。我们开发了用于测量在一个蛋白的水平在中心体中固定的细胞来自不同样品的相对变化，在细胞周期的不同阶段，例如，或用各种试剂处理后的定量荧光显微术测定。这个测定的原理在于测量对应于蛋白质在一个小区域的背景校正的荧光强度，和正常化了的测量对相同的对不选择的实验中c而变化的另一蛋白质ondition。利用该测定在用BrdU脉冲和追策略学习未扰动细胞周期的组合，我们已定量具体地说在中心体验证我们最近的观察结果VDAC3的中心体池在细胞周期中被调节在中心体，可能是由蛋白酶体介导的降解。
Introduction
中心体由一对中心粒由pericentriolar材料（PCM）所包围的。为在哺乳动物细胞中主要的微管组织中心（MTOCs），中心体作为有丝分裂纺锤体的两极在分裂的细胞，从而有助于维持基因组完整性1。在静止期细胞（ 例如 ，在G0期）时，中心体，即母中心粒的两个中心粒中的一个，被转换成一个基体组装在主纤毛，感官细胞器从细胞表面2突出出来。一旦细胞重新进入细胞周期，原发性纤毛被拆卸并且每个中心粒指挥一个procentriole在其近端逐渐伸长，以形成一个成熟的中心粒3的组装。在S期的开始，一个侧手翻状结构，它提供了9倍对称性的中心粒形成每个现有中心粒的表面上，将成为各procentriole的基础。 SAS6吨帽子是必不可少的中心粒组件被招募来形成手翻4-6的中心。其他中心粒蛋白，然后组装到侧手翻在一个高度管制，近端远端的方式7。经过精确完成中心粒的复制，细胞聚集更多的pericentriolar材料由G2期8月底前建立两个功能中心体。在除了核心部件中心粒9-11，其它几种蛋白质，包括蛋白激酶，磷酸酶，蛋白伴侣，脚手架元件，膜相关蛋白和降解机械与中心粒，基体和PCM在细胞周期12-16的不同的时间相关联。它经常被注意的是，许多蛋白质的中心体的水平在时间上由中心体定位的机制和/或蛋白酶体降解在中心体调节。重要的是，在几个蛋白质如PLK4，MPS1，SAS6和CP110 a的中心体水平的波动吨的不同点的细胞周期似乎是至关重要的调节中心粒装配5,17-22，和在MPS1的防止这个中心体的降解的情况下会导致过量中心粒19的形成。另一方面，相对于细胞溶质池几种蛋白质的中心体部分较少不稳定。例如的siRNA介导的下调中金2（Cetn2）导致了在中心粒蛋白水平仅温和下降，尽管大大降低了整个细胞水平的23。因此，关键是要测量在中心体的变化中心体蛋白的水平，而不是评估其中心体特定的功能时，测定全细胞蛋白质的水平。
在这项研究中，我们开发了使用间接免疫荧光（IIF）量化蛋白质在中心体的相对水平的测定法。该测定被显影尤其来分析细胞，来自不同样品因此不能在同一时间被成像。这些样品可以是用不同的试剂（ 即 ，药物与对照）中，在不同的时间点收集经处理的细胞（ 即 ，脉搏与追），或者是在细胞周期的不同阶段。这个测定的原理在于测量对应于蛋白质在一个小区域和正常化针对同一该值对于另一种蛋白质，其水平所选择的实验条件下不发生变化的背景校正的荧光强度。在中心体生物学几项研究最近使用的各种定量显微技术，在这两种活的或固定细胞，以确定候选蛋白24-27的中心体特定的函数。类似测定法，本发明的技术还可以测量测试蛋白的背景校正的荧光强度。然而，在该测定法使用内标列入正常化将可能提供更多的准确性和信心在分析两种不同的样本是在两个不同的盖玻片。此外，除了在研究中心体蛋白质水平，用小的调整这种方法可以应用到的实验条件多样化或其它细胞位点。
在这里，我们结合定量检测显微镜用的BrdU脉冲追踪策略，比较不同的细胞周期阶段的细胞。代替使用标准的细胞周期停滞和释放技术，研究各种细胞周期的时间点，以异步方式生长的细胞的孵育用BrdU在S期标记细胞，标记的细胞追逐不同的时间（通常为4-6小时）。大多数标记的细胞将在脉冲后立即处于S期。追逐的长度被选择为使得追后，标记细胞将在晚S期，G2或有丝分裂，其可以通过形态特征中心体相对于NUC如 – 位置进行验证雷，中心体之间的距离，染色体等的缩合因此，追逐的长度取决于为S，G2和特定细胞类型的M期的平均持续时间。由于这种方法避免了细胞周期抑制剂，如羟基脲，阿非迪霉素，诺考达唑等 ，其允许更生理学相关细胞周期分析。
因此，我们在这里表明，单独的定量荧光显微术测定中，或在用BrdU脉冲追踪试验组合，是一种简单而功能强大的技术中的未扰动的细胞周期，以精确地测量在候选蛋白质的中心体水平的相对变化。我们测量VDAC3，我们在中心体最近确定除了线粒体16,28的蛋白质的中心体的水平，使用这些测定。在这里所得到的结果验证我们之前的看法，即VDAC3的中心体游泳池被降解调节，也各有不同的细胞周期dependen吨方式16中 ，还证实了该方法的适用性。
Protocol
Representative Results
Discussion
在细胞生物学定量显微镜通常与活细胞成像试验，如荧光共振能量转移（FRET），荧光恢复漂白相关（FRAP） 等。然而，后有细胞生物学家的例子越来越多开发不同的定量检测显微镜对固定细胞近年来27,34-36。重要的是，了解中心体生物学的进步往往需要的蛋白质，其中心体池可能会受到不同的管制比其他池的中心体特异功能的理解。因此，我们开发了一个定量的IIF分析特异性分析的…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by a National Institutes of Health grant (GM77311) and a seed grant from The Ohio Cancer Research Associates (to H.A.F.). SM was partially supported by an Up on the Roof fellowship from the Human Cancer Genetics Program of The Ohio State University Comprehensive Cancer Center.
Materials
| Name | Company | Catalog number | Comments |
| Fibronectin | Sigma | F8141 | Stock of 1 mg/ml in water |
| DMSO | Sigma | D2650 | |
| MG115 | Sigma | SCP0005 | Stock of 10 mM in DMSO |
| BrdU | Sigma | B5002 | Stock of 10 mM in DMSO |
| Anti-g-tubulin (mouse monoclonal, clone GTU 88) | Sigma | T 6557 | 1:200 in IIF blocking buffer |
| Anti-VDAC3 (rabbit polyclonal) | Aviva Systems Biology | ARP35180-P050 | 1:50 in IIF blocking buffer, 1:1000 in WB blocking buffer |
| Anti-Sas6 (mouse monoclonal) | Santa cruz biotechnology | sc-81431 | 1:100 in IIF blocking buffer |
| Anti-Cep135 (rabbit polyclonal) | Abcam | ab-75005 | 1:500 in IIF blocking buffer |
| Anti-BrdU (rat monoclonal) | Abcam | ab6326 | 1:250 in IIF blocking buffer |
| Alexa Fluor 350 Goat Anti-Rat IgG (H+L) | Life technologies | A21093 | 1:200 in IIF blocking buffer |
| Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L) Antibody | Life technologies | A21202 | 1:1000 in IIF blocking buffer |
| Alexa Fluor 594 Donkey Anti-Mouse IgG (H+L) Antibody | Life technologies | A21203 | 1:1000 in IIF blocking buffer |
| Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L) Antibody | Life technologies | A21206 | 1:1000 in IIF blocking buffer |
| Alexa Fluor 594 Donkey Anti-Rabbit IgG (H+L) Antibody | Life technologies | A21207 | 1:1000 in IIF blocking buffer |
| Anti-g-tubulin (rabbit polyclonal) | Sigma | T5192 | 1:1000 in WB blocking buffer |
| Anti-a-tubulin (mouse monoclonal, DM1A) | Sigma | T9026 | 1:20000 in WB blocking buffer |
| Alexa Fluor 680 Donkey Anti-Rabbit IgG (H+L) | Life technologies | A10043 | 1:10000 in WB blocking buffer |
| Mouse IgG (H&L) Antibody IRDye800CW Conjugated | Rockland antibodies | 610-731-002 | 1:10000 in WB blocking buffer |
| SlowFade Gold Antifade Reagent | Life technologies | S36936 | Mounting media |
| Round coverslips 12CIR.-1 | Fisherbrand | 12-545-80 | |
| Olympus IX-81 microscope | Olympus | ||
| Retiga ExiFAST 1394 IR camera | QImaging | 32-0082B-238 | |
| 100X Plan Apo oil immersion objective | Olympus | 1.4 numerical aperture | |
| Slidebook software package | Intelligent Imaging Innovations | ||
| Odyssey IR Imaging System | Li-cor Biosciences | ||
| Bicinchoninic acid (BCA) assay | Thermo Scientific | 23227 | |
| U-MNU2 Narrow UV cube | Olympus | U-M622 | Filter |
| U-MNU2 Narrow Blue cube | Olympus | U-M643 | Filter |
| U-MNU2 Narrow Green cube | Olympus | U-M663 | Filter |
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