Name: use of two dimensional semi-denaturing detergent agarose gel electrophoresis to confirm size heterogeneity of amyloid or amyloid-like fibers
Uploaded: 2018-04-26T14:30:00
Description: Watch this Scientific Journal Video about Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers at JoVE.com

这项工作得到了何善衡奖学金的支持。从 NIH/NCATS (TL1TR001104), 韦尔奇基金会赠款 (I-1827) 和研究院 (RGM120502A) R01 赠款 z.w.z. W. 是弗吉尼亚默奇森克姆学者在医学研究和癌症预防和研究所得克萨斯学者 (R1222)。

1. 准备样品
<ol><li>培养 2 x 106淀粉样蛋白, 在10厘米组织培养皿中产生 HT-29 结肠癌细胞, 10 毫升的 Dulbecco 修饰鹰培养基 (DMEM) 含有10% 胎牛血清和青霉素-链霉素。培养细胞过夜在37°c 孵化器与 5% CO2。<ol>
<li>细胞生长到80% 融合后, 用10毫升磷酸盐缓冲的生理盐水 (PBS) 冲洗细胞。加入3毫升胰蛋白酶溶液, 在37摄氏度孵育3分钟。</li>
		<li>细胞完全脱离皿后, 加入10毫升培养基, 将10毫升吸?...

<ol>
	<li>Ross, C. A., Poirier, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+aggregation+and+neurodegenerative+disease.">Protein aggregation and neurodegenerative disease.</a> Nature medicine. 10, 10-17 (2004).</li><li>Hou, F., 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=MAVS+forms+functional+prion-like+aggregates+to+activate+and+propagate+antiviral+innate+immune+response.">MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.</a> Cell. 146, 448-461 (2011).</li><li>Li, 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=The+RIP1/RIP3+necrosome+forms+a+functional+amyloid+signaling+complex+required+for+programmed+necrosis.">The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis.</a> Cell. 150, 339-350 (2012).</li><li>Liu, S., 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=MLKL+forms+disulfide+bond-dependent+amyloid-like+polymers+to+induce+necroptosis.">MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 114, 7450-7459 (2017).</li><li>Uptain, S. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+as+protein-based+genetic+elements.">Prions as protein-based genetic elements.</a> Annual review of microbiology. 56, 703-741 (2002).</li><li>Halfmann, R., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+amyloid+aggregation+by+Semi-Denaturing+Detergent-Agarose+Gel+Electrophoresis.">Screening for amyloid aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis.</a> J Vis Exp. , (2008).</li><li>Sipe, J. D., 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=Nomenclature+2014:+Amyloid+fibril+proteins+and+clinical+classification+of+the+amyloidosis.">Nomenclature 2014: Amyloid fibril proteins and clinical classification of the amyloidosis.</a> Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis. 21, 221-224 (2014).</li><li>Eisenberg, D. S., Sawaya, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Studies+of+Amyloid+Proteins+at+the+Molecular+Level.">Structural Studies of Amyloid Proteins at the Molecular Level.</a> Annual review of biochemistry. 86, 69-95 (2017).</li><li>Bagriantsev, S. N., Kushnirov, V. V., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 412, 33-48 (2006).</li><li>Sun, L., 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=Mixed+lineage+kinase+domain-like+protein+mediates+necrosis+signaling+downstream+of+RIP3+kinase.">Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.</a> Cell. 148, 213-227 (2012).</li><li>He, S., 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=Receptor+interacting+protein+kinase-3+determines+cellular+necrotic+response+to+TNF-alpha.">Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.</a> Cell. 137, 1100-1111 (2009).</li></ol>

2D SDD 年龄的最关键的方面是电泳条件是相同的第一和第二个维度。在 45° (表明没有离解或退化发生) 的情况下, 检测到一条锋利的对角线的能力取决于在两个电泳中以相同方式迁移的纤维。使用不同的条件, 例如改变电压或运行时间的长短, 将会掩盖这些结果。同样, 与传统的 1D SDD 年龄的情况一样, 煮沸的样品应该避免, 因为这将扰乱淀粉样蛋白和淀粉样的纤维。与所有转移的情况一样, 应注意避?...

由于蛋白质错误折叠, 淀粉样纤维的形成早已被人们所熟知, 它在阿尔茨海默病、帕金森病、亨廷顿病等病理状况中起到了1的作用.最近, 淀粉样蛋白或淀粉样纤维的形成已被证明是人类信号通路的一部分, 包括在抗病毒先天免疫应答中2和 necroptosis3,4, 和在较低的有机体如酵母5,6。因此, 在实验室中检测这些纤维的能力是很重要的。目前, 有三种主要的方法来检测淀粉样蛋白和淀粉样的纤维: 使用染料, EM 和 SDD 年龄。
使用染料, 如刚果红或 Thioflavin T, 提供了快速和容易检测使用显微镜或光谱学7的优势。然而, 在刚果红的情况下, 显微镜下的检测不提供任何蛋白质组成纤维或纤维大小的特异性。同样, 使用光谱学检测刚果红或 Thioflavin T 结合蛋白复合物只提供了积极或消极的结果。
EM 提供了有关纤维的存在的确凿证据, 以及关于光纤长度和直径8的定量信息。然而, 这种方法需要非常严格的净化。此外, EM 是一种专门的技术, 使用昂贵的设备。
SDD 年龄已被用来检测抗 SDS 的巨型道尔顿蛋白复合物, 包括淀粉样蛋白或淀粉样的纤维。它提供了许多优点。首先, 它不需要对纤维进行纯化, 并且很容易请执行 9.其次, 它提供了有关纤维大小的定性信息, 包括纤维 heterogenicity 的相对大小和数量。最后, 由于西方印迹可以在电泳后进行, 所以很容易检测出有抗体的任何蛋白质的存在, 虽然应该指出, 由于 SDD 年龄是半变性, 一些表位可能仍然隐蔽通过抗体使得检测复杂化。
最近, 据报道, 受体相互作用蛋白激酶 1 (RIPK1) 和 3 (RIPK3) 形成淀粉样纤维作为信号平台在 necroptosis 期间, 一个程序性的形式坏死3。在研究这些纤维时, 我们的实验室发现另一种 necroptosis 相关的蛋白质, 混合谱系激酶域样 (MLKL), 也形成淀粉样的纤维4。然而, 在 SDD 年龄的检查, MLKL 纤维的大小似乎有别于 RIPK1 和 RIPK3 纤维, 这似乎彼此相同 (图 1)。这是意外的, 因为众所周知, MLKL 绑定到 RIPK1/RIPK3, 形成称为 necrosome10的 necroptosis 信号复合体。
至少有两种解释。首先, 两个完全不同的淀粉样的纤维可能形成在 necroptosis, 一个含有 RIPK1/RIPK3 和其他含有 MLKL。其次, 在 necroptosis 期间, 只有一种含有 RIPK1/RIPK3/MLKL 的淀粉样纤维可以形成, 但 MLKL 与其他蛋白质的关联性较弱, 在 SDD 时代离解。
为了解决这个问题, 我们建议执行一个二维 (2D) SDD 年龄。在第一和第二维电泳中, SDD 稳定的淀粉样蛋白或淀粉样蛋白纤维将具有相同的迁移模式。这将是可检测的, 将蛋白质转移到膜, 并进行了一个西方的印迹。稳定的纤维会呈现尖锐的对角线图案。任何偏离这一点将表明, 纤维发生变化, 由于 SDS 电泳。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >gel electrophoresis unit</td>
			<td >Fisher</td>
			<td >HE99XPRO</td>
			<td >appratus for gel running.</td>
		</tr>
		<tr >
			<td >agrose</td>
			<td >VWR</td>
			<td >97062-250</td>
			<td>For agarose gel.</td>
		</tr>
		<tr >
			<td >paper tower</td>
			<td >Fisher</td>
			<td >19-120-2484</td>
			<td>for transfering</td>
		</tr>
		<tr >
			<td >filter paper</td>
			<td >VWR</td>
			<td >21427-386</td>
			<td>for transfering</td>
		</tr>
		<tr >
			<td >PVDF membrane</td>
			<td >Bio-Rad</td>
			<td >162-0177</td>
			<td>for transfering</td>
		</tr>
		<tr >
			<td >blocking milk powder</td>
			<td >Bio-Rad</td>
			<td >1706404XTU</td>
			<td>for blocking in Western blot</td>
		</tr>
		<tr >
			<td >MLKL antibody</td>
			<td >Genetex</td>
			<td >GTX107538</td>
			<td>rabbit anti-MLKL antibody</td>
		</tr>
		<tr >
			<td >RIPK1 antibody</td>
			<td >BD Biosciences</td>
			<td >610459</td>
			<td>mouse anti-RIPK1 antibody</td>
		</tr>
		<tr >
			<td >RIPK3 antibody</td>
			<td >gift from Dr. Xiaodong Wang</td>
			<td ></td>
			<td>rabbit anti-RIPK3. See refenence 11.</td>
		</tr>
		<tr >
			<td >anti-Rabbit-HRP</td>
			<td >Sigma</td>
			<td >A6154</td>
			<td>secondary antibody</td>
		</tr>
		<tr >
			<td >ECL</td>
			<td >Fisher</td>
			<td >WBKLS0500</td>
			<td>Western chemiluminescent HRP substrate</td>
		</tr>
		<tr >
			<td >X-ray film</td>
			<td >Fisher</td>
			<td >F-BX810</td>
			<td>for Western blot result</td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td >Sigma</td>
			<td >D6429</td>
			<td>for cell culture</td>
		</tr>
		<tr >
			<td >fetal bovine serum</td>
			<td >Sigma</td>
			<td >F4135</td>
			<td>for cell culture</td>
		</tr>
		<tr >
			<td >penicilin-streptomycin</td>
			<td >Sigma</td>
			<td >P4333</td>
			<td>for cell culture</td>
		</tr>
		<tr >
			<td >Trypsin solution</td>
			<td >Sigma</td>
			<td >T4049</td>
			<td>for cell culture</td>
		</tr>
		<tr >
			<td >PBS for tissue culture</td>
			<td >Sigma</td>
			<td >D8662</td>
			<td>for cell culture</td>
		</tr>
		<tr >
			<td >recombinant TNF</td>
			<td >made in our lab</td>
			<td ></td>
			<td>for inducing necroptosis. See reference 11.</td>
		</tr>
		<tr >
			<td >smac-mimetic</td>
			<td >gift from Dr. Xiaodong Wang</td>
			<td ></td>
			<td>for inducing necroptosis. See reference 11.</td>
		</tr>
		<tr >
			<td >ZVAD-FMK</td>
			<td >ApexBio</td>
			<td >A1902</td>
			<td>for inducing necroptosis. See reference 11.</td>
		</tr>
		<tr >
			<td >Cell Counter</td>
			<td >Bio-Rad</td>
			<td>1450102</td>
			<td>Model TC20; for counting cells</td>
		</tr>
		<tr >
			<td >Pierce&#8482; BCA Protein Assay Kit</td>
			<td >Thermo Scientific</td>
			<td >23225</td>
			<td>for measuring protein concentration in cell lysates</td>
		</tr>
		<tr >
			<td >Cell lifter</td>
			<td >Fisher</td>
			<td >07-200-364</td>
			<td>to remove cells from dish</td>
		</tr>
		<tr >
			<td >Lysis Buffer (1 L)</td>
			<td></td>
			<td></td>
			<td >20 mL 1 M Tris pH 7.4 
			10 mL glycerol 
			30 mL 5 M NaCl 
			840 mL ddH2O 
			10 mL Triton-X100 
			(protease and phosphates inhibitors as desired)</td>
		</tr>
		<tr >
			<td >10X TAE (1 L)</td>
			<td></td>
			<td></td>
			<td >48.4 g Tris base 
			11.42 mL glacial acetic acid 
			20 mL 0.5M EDTA pH 8 
			ddH20 to 1 L</td>
		</tr>
		<tr >
			<td >4X SDD-AGE loading buffer (50 mL)</td>
			<td></td>
			<td></td>
			<td >5 mL 10X TAE 
			10 mL glycerol 
			4 mL 20% SDS 
			0.5 mL 10% bromophenol blue 
			31 mL ddH2O</td>
		</tr>
		<tr >
			<td >TBS Transfer Buffer (1 L)</td>
			<td></td>
			<td></td>
			<td >20 mL 1 M Tris pH 7.4 
			30 mL 5 M NaCl 
			950 mL ddH2O</td>
		</tr>
		<tr >
			<td >PBST Wash Buffer (1 L)</td>
			<td></td>
			<td></td>
			<td >100 mL 10xPBS 
			800 mL ddH2O 
			1 mL Tween20</td>
		</tr>
		<tr >
			<td >10X PBS (10 L)</td>
			<td></td>
			<td></td>
			<td >800 g NaCl 
			20 g KCl 
			144 g Na2HPO4&#183;2H2O 
			24 g KH2PO4 
			add ddH2O to 10 L</td>
		</tr>
		</tbody>
</table>

use of two dimensional semi-denaturing detergent agarose gel electrophoresis to confirm size heterogeneity of amyloid or amyloid-like fibers

淀粉样蛋白或像淀粉样的纤维已经与许多人类疾病相关, 现在被发现对许多信号通路是重要的。在不同的实验条件下, 能够很容易地检测出这些纤维的形成, 对于了解它们的潜在功能是至关重要的。许多方法已被用来检测纤维, 但没有一些缺点。例如, 电子显微镜 (EM), 或染色与刚果红或 Thioflavin T 往往需要纯化的纤维。另一方面, 半变性洗涤剂琼脂糖凝胶电泳 (SDD 年龄) 允许检测的 SDS 耐药淀粉样纤维在细胞提取物没有纯化。另外, 它允许比较纤维的大小区别。更重要的是, 它可以用来识别纤维内的特定蛋白质由西方印迹。它的耗时更少, 更容易被更多的实验室所访问。SDD 年龄的结果往往表现出不同程度的异质性。它提出了一个问题, 即非异质性的一部分是由蛋白质复合体在电泳过程中的分离而产生的。由于这个原因, 我们使用了 SDD 年龄的第二个维度来确定 SDD 年龄所看到的大小异质性是否真的是纤维异质性在体内的结果, 而不是由于某些蛋白质的降解或离解而导致电泳。这种方法可以快速、定性地确认淀粉样蛋白或淀粉样蛋白纤维在 SDD 年龄过程中没有部分游离。

在 necroptosis 诱导, RIPK1 和 RIPK3 显示几乎相同的淀粉样蛋白样的模式 (车道2和 4,图 1)。然而, MLKL 纤维更异质, 似乎小于 RIPK1/RIPK3 纤维 (6 车道,图 1)。这促使我们开发了 2D SDD 的方法, 以解决 MLKL 是否形成 RIPK1/RIPK3-independent 纤维或 MLKL 简单离解在 RIPK1/RIPK3/MLKL 年龄的大 SDD 纤维的可能性。
<p class="jove_content" fo:keep-together.within-page="...

Watch this Scientific Journal Video about Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers at JoVE.com

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

二维半变性洗涤剂琼脂糖凝胶电泳法确定淀粉样或淀粉样纤维的粒度异质性

在这里, 我们使用二维半变性琼脂糖凝胶电泳, 以确认存在的异质淀粉样纤维的异构大小, 并排除了大小异质性是由于淀粉样纤维在凝胶中离解的可能性运行过程。

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