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毫升吸管的细胞转移到15毫升圆锥管。在室温下将细胞以 1000 x g 为3分钟离心。吸入培养基, 并用重悬5毫升培养基中的细胞, 并用细胞计数器计数细胞。板 2 x 106单元格中每两个10厘米的菜肴。</li>
		<li>允许细胞在一夜之间坚持和恢复在37°c 孵化器与 5% CO2。对一道菜进行治疗, 以诱导 20 amyloids 肿瘤坏死因子α (TNF α), 100 nM Smac 模拟, 20 µM 泛 caspase 抑制剂 Z-(芬兰马克11 的形成。组合缩写为慈。把另一盘车当作控制品对待。</li>
	</ol>
</li>
	<li>在适当的时间长度以后, 通常 6 h, 收获细胞裂解。<ol>
<li>用塑料刮刀刮掉盘子上的细胞, 然后用10毫升的吸管将其转移到15毫升圆锥管中。离心细胞在 1000 x g 3 分钟在4摄氏度。</li>
		<li>2次通过 resuspending 在10毫升的冰冷 PBS 和离心在 1000 x g 3 分钟, 在4摄氏度清洗细胞。吸入 PBS 溶液。 注: 这个过程可以在这里暂停冻结细胞颗粒在液氮和储存 at#˗80°c 长达1月。</li>
		<li>将细胞颗粒转移到1.5 毫升的离心管上, 在0.3 毫升的裂解缓冲液中孵育30分钟冰。离心机在 2万 x g 15 分钟在4摄氏度。上清是整个细胞裂解。 注意: 这个过程可以在这里暂停, 将样品储存在-20 摄氏度, 长达几个月。</li>
		<li>根据制造商的协议测量蛋白质浓度的布拉德福德化验。添加 4x SDD 龄加载缓冲器, 准备20µL 的3µg/µL 样品, 室温孵育10分钟。</li>
	</ol>
</li>
</ol>2. 准备和运行凝胶
<ol><li>在玻璃烧杯中添加2克琼脂糖到200毫升的1x 三醋酸盐缓冲液中, 在微波炉中加热以融化琼脂糖。添加1毫升 20% sds, 最终浓度为 0.1% sds。仔细地旋转混合。注意不要在 SDS 添加后产生气泡。</li>
	<li>将琼脂糖溶液倒入15厘米 x 14 厘米的凝胶板中。使用1毫升吸管消除任何气泡。在顶部放一把20好梳子。</li>
	<li>第一维: 吸管60µg 全细胞裂解在极右车道。在 60 V 左右运行凝胶约 4 h (直到染料前面是关于¾通过凝胶) 使用泰含 0.1% SDS 作为运行缓冲。</li>
	<li>第二维: 小心旋转凝胶90°逆时针方向 (图 2A)。用60伏的凝胶大约4小时。 注: 一般运行条件为4伏/厘米凝胶长度。重要的是, 在第一和第二维度上, 运行条件是完全相同的。</li>
</ol>3. 转让
<ol><li>使用毛细管转移方法将蛋白质转移到聚偏聚氟 (PVDF) 膜 (图 2B)。<ol>
<li>将500毫升的传输缓冲器添加到大约20厘米 x 20 厘米的容器中。在容器旁边制作一叠5厘米高的纸巾。堆栈顶部的表面积必须大于凝胶尺寸。</li>
		<li>将14厘米 x 15 厘米的滤纸浸泡在传送缓冲器中, 放在纸巾栈的顶部。注意不要在纸上放皱纹或气泡。用另一张滤纸重复。</li>
		<li>激活 14 cm x 15 cm PVDF 膜在甲醇三十年代然后在过滤器纸上把它层起来小心使用滚筒去除所有气泡。用转移缓冲器冲洗凝胶并将其层在膜上, 再滚出任何气泡。</li>
		<li>覆盖最靠近容器的纸巾的边缘与塑料包装。在下一步施工的纸桥不与纸巾栈直接接触是很重要的。</li>
		<li>在传输缓冲器中浸泡一块15厘米 x 35 厘米的滤纸, 然后将其放在一个端盖上, 另一端位于传输缓冲器的容器中。与另一座桥重复。</li>
		<li>用塑料包装把容器盖住, 在室温下过夜。</li>
	</ol>
</li>
</ol>4. 西方印迹检测
<ol><li>用50毫升的 PBS 冲洗膜, 其中 0.05% Tween-20 (PBST) 在20厘米 x 20 厘米的容器中。在 PBST 中加入20毫升5% 牛奶。在室温下晃动30分钟以阻挡。</li>
	<li>吸管10µL 兔抗 MLKL 抗体成20毫升5% 牛奶在 PBST 和添加到容器。在摇杆上孵化过夜在4摄氏度。</li>
	<li>用20毫升的 PBST 冲洗膜5分钟, 重复5次。</li>
	<li>吸管4µL 的抗兔 HRP 到20毫升5% 牛奶在 PBST 和添加到容器。在2小时的室温下在摇杆上孵化。</li>
	<li>用20毫升的 PBST 冲洗膜5分钟, 重复5次。</li>
	<li>添加增强化学发光基底 (ECL), 并根据制造商的协议暴露在 X 射线胶片。</li>
</ol>

<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>

作者声明没有利益冲突。

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。
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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。

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

use-two-dimensional-semi-denaturing-detergent-agarose-gel

Research

JoVE Journal

Biochemistry

9.7K 次观看.  UT Southwestern Medical Center. 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。

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

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

使用非变性聚丙烯酰胺凝胶电泳人的MxA多亚基蛋白复合物的表征

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

科研

生物化学

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

蛋白质tRNA琼脂糖凝胶延迟测定分析<em&gt;ñ</em<sup&gt; 6</sup&gt; - 磺酰氨基甲酰腺苷TcdA功能

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

人死后脑中洗涤剂不溶性蛋白的富集

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

水平凝胶电泳用于增强蛋白质 -  RNA复合物的检测

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

This protocol describes the characterization of DNA polymerase synthesis of modified DNA through observation of changes to near-infrared fluorescently labeled DNA using gel electrophoresis and gel imaging. Acrylamide gels are used for high resolution imaging of the separation of short nucleic acids, which migrate at different rates depending on size.

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

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 ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

毛细管电泳与亲和毛细管电泳对金属离子 AtHIRD11 固有紊乱与结合的分析

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

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

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