人死后脑中洗涤剂不溶性蛋白的富集
概要
本文介绍了一种从人死后大脑中浓缩洗涤剂不溶性蛋白集料的简化分馏方法。
Abstract
在本研究中, 我们描述了一个缩写的单步分馏协议, 以丰富的洗涤剂不溶性蛋白聚集从人死后大脑。离子洗涤剂 n-十二烷基肌 (sarkosyl) 有效地 solubilizes 本机折叠蛋白质在脑组织允许丰富的洗涤剂不溶性蛋白聚集从广泛的神经退行性 proteinopathies, 如阿尔茨海默病 (AD), 帕金森氏症和肌萎缩侧索硬化症, 和朊病毒疾病。人的控制和 AD 死后的脑组织均匀化和沉淀的离心存在的 sarkosyl 丰富的洗涤剂不溶性蛋白骨料, 包括病理磷酸化 tau, 纤维的核心组成部分在广告中缠结。西部印迹显示了聚合磷酸化 tau 和洗涤剂可溶性蛋白, 早期 Endosome 抗原 1 (EEA1) 在控制和 AD 脑的差异溶解度。蛋白质组分析还显示, 与对照组相比, 在 AD 脑 sarkosyl 不溶性的片段中, β淀粉样蛋白 (a)、tau、snRNP70 (U1-70K) 和载脂蛋白 E (脂蛋白) 的富集与以前的组织分馏策略相一致.因此, 这一简单的浓缩协议是理想的广泛的实验应用范围从西方印迹和功能蛋白 co-aggregation 化验, 以大规模谱蛋白质组学。
Introduction
神经退行性疾病, 如阿尔茨海默氏病 (AD)、帕金森氏病 (PD)、亨廷顿氏病 (HD)、肌萎缩侧索硬化症 (ALS), 以及与之密切相关的朊病毒疾病, proteinopathies 的特点是逐渐积累洗涤剂-不溶性蛋白质聚集在大脑中伴随着认知能力的下降。1,2这种共同的病理特征被认为在这些神经退行性疾病的病因和病理生理学中起着中心作用。2这些聚合体通常由聚合淀粉样纤维组成, 它们由错误蛋白的重复单位构成, 其表现为跨β结构。1,2,3,4生化, 淀粉样蛋白聚合体对化学或热变性和增溶具有很强的抗性,3在传统生化技术中对其纯化、分析和研究提出了独特的挑战。2,5,6,7,8,9,10,11不难的是, 不溶性的蛋白质组分一直是许多研究神经退行性疾病的病理生理学的焦点, 涉及错误蛋白的积累。6,12,13,14
生化分馏技术经常被用来丰富从死后脑匀的洗涤剂不溶性分数。6,12,13,14最常见的方法之一是对组织匀进行顺序提取, 并增加严格的缓冲剂和洗涤剂, 然后离心分解可溶性和不溶性组分。通常使用的顺序分馏协议6,14涉及在无洗涤剂低盐 (LS) 缓冲液中的冷冻组织样品的均一性, 然后依次提取含有高盐, 非离子洗涤剂, 高蔗糖, 离子洗涤剂, 最后 chaotropes 像尿素的缓冲器。6,14此类顺序分馏协议的一个明显的缺点是完成它们所需的大量时间和人工承诺。包括均匀化和离心, 一个典型的五步分馏协议可能需要几个小时甚至几天的时间来完成。4,6,7,10,15,16,17,18此外, 由于许多病理蛋白聚集物仍然不溶于所有, 但最苛刻的条件19,20大多数生成的分数都是有限的值。因此, 使用高盐浓度和非离子洗涤剂的不太严格的分馏步骤基本上是多余的。
以往的研究表明, 离子洗涤剂 n-十二烷基肌 (sarkosyl) 是一个很好的候选的简化一步洗涤剂分馏协议。5,6,12,13,14,21,22,23作为一种变性洗涤剂, sarkosyl 是严格的, 足以溶解在大脑中的绝大多数本机折叠的蛋白质, 没有磷错误蛋白聚合组成β-淀粉样 (a),6,11磷酸化 tau (pTau),6焦油 DNA 结合蛋白 43 (TDP-43),14 α-核,12,13病,23或 U1 小核均一 (U1 snRNPs), 如 U1-70K。5,21,22由于 sarkosyl 比无处不在的阴离子洗涤剂十二烷基硫酸钠 (sds) 的严格, 它保留了不太坚固的聚形式的错误蛋白骨料, 无法抵御 sds 治疗。9
以前, 我们描述了一个缩写的洗涤剂分馏协议, 取得的结果可与更费力的顺序分馏方法相媲美。5通过省略较不严格的分馏步骤, 我们得以开发出一种简便的单步分馏协议, 用于丰富死后人脑中的洗涤剂不溶性蛋白聚集物。5本文所述的详细协议非常适合于从西方印迹和大量谱蛋白质组学到功能性蛋白质折叠和聚合播种试验等一系列实验应用。5,6,21
Protocol
Representative Results
Discussion
在此, 我们介绍并讨论了一个简化的单步洗涤剂分馏协议, 适用于广泛的各种实验应用范围从质量谱蛋白质组学分析到功能蛋白折叠测定和西部印迹。5,6,7,10这种方法可能是最有效的, 当用于研究神经退行性 proteinopathies, 如阿尔茨海默氏症, ALS, 亨廷顿氏症, 朊病毒疾病和各种 tauopathies。与原来的五步顺?…
開示
The authors have nothing to disclose.
Acknowledgements
作者感谢 Drs 和利维, 埃默里神经病学系, 提供有用的意见和建议。这项工作的部分资金来自加速医学合作基金 (U01AG046161-02), 埃默里阿尔茨海默病研究中心 (P50AG025688) 和国家老龄补助金研究所 (R01AG053960-01) 运输这项研究也得到了部分的支持, 神经病核心的埃默里神经科学一核心设施补助金, P30NS055077。
Materials
| Protease and phosphatase inhibitor cocktail, EDTA-free (100X) | Thermo Fisher | 78441 | protease & phosphatase inhibitor cocktail |
| Sonic Dismembrator System (ultrasonicator) | Fisher Scientific | FB505110 | microtip ultrasonicator |
| Optimax TLX Ultracentrifuge | Beckman Coulter | 361545 | refrigerated ultracentrifuge |
| TLA120.1 rotor | Beckman Coulter | 362224 | ultracentrifuge rotor |
| 500 ul (8x34mm) polycarbonate tubes, thickwall | Beckman Coulter | 343776 | ultracentrifuge tubes for TLA120.1 rotor |
| 4X SDS sample buffer | Home-made | N/A | SDS-PAGE |
| TCEP solution, neutral pH | Thermo Fisher | 77720 | reducing agent |
| (TBS) blocking buffer | Thermo Fisher | 37542 | blocking buffer |
| (TBS) blocking buffer + 0.05% Tween 20 | Thermo Fisher | 37543 | blocking buffer and antibody diluent |
| 4-12% Bolt Bis-Tris Plus gels, 10-well | Thermo Fisher | NW04120BOX | precast SDS-PAGE gels |
| MES SDS Running Buffer (20X) | Thermo Fisher | B0002 | SDS-PAGE running buffer |
| N-Lauroylsarcosine sodium salt (sarkosyl) | Sigma Aldrich | L5777-50G | detergent |
| 1.5 ml polypropylene Pellet Pestle | Kimble Chase | 749521-1500 | homogenization tool |
| cordless motor for Pellet Pestle | Kimble Chase | 749540-0000 | homogenization tool |
| Anti-Tau-2 (pan tau) antibody | Chemicon | MAB375 | antibodies |
| Anti-phospho-threonine 231 Tau antibody | Millipore | MAB5450 | antibodies |
| Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8) | Thermo Fisher | MN1020 | antibodies |
| Anti-early endosome antigen 1 (EEA1) antibody | abcam | ab2900 | antibodies |
| Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody | Thermo Fisher | A21058 | antibodies |
| Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody | Thermo Fisher | A11374 | antibodies |
| iBlot2 Dry Blotting System | Thermo Fisher | IB21001 | Gel transfer |
| iBlot2 Transfer Stacks, Nitrocellulose, mini | Thermo Fisher | IB23002 | Gel transfer |
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