这项工作得到了基金发展 Científico y Tecnológico (科技), 智利, 项目 11130263 (cw), 项目会 + NERC + 方案 de Colaboración 国际 PCI-PII20150073 (对 cw) 和 U inicia 从 Vicerrectoría de智利研究大学 (对 CW)。
感谢纳兰霍-帕尔马在这个项目的初始阶段的帮助, Dr. 巴布尔和迭基罗加-罗杰为有益的讨论, 罗伯特 Lesch 修订手稿和总署 de extensión Facultad 德 Ciencias Químicas yFarmacéuticas 来自智利大学。也感谢马塞洛佩雷斯的编辑视频和尼尔史蒂文斯为纠正文本和沃尔华提供的声音-结束。

1. 凝胶的制备和运行
<ol><li>用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (sds 页) 在理查德·山姆布鲁克和罗素14中发现的不变性12% 聚丙烯酰胺凝胶的配方, 但使用水代替 sds。<ol>
<li>要准备5毫升的溶解凝胶, 使用1.7 毫升蒸馏水, 2 毫升丙烯酰胺/双丙烯酰胺 (30/0.8% 瓦特/五), 1.3 毫升1.5 米的 pH 8.8, 0.05 毫升的10% 硫酸铵, 和0.002 毫升的 TEMED。</li>
		<li>准备2毫升的堆叠凝胶使用1.5 毫升水, 0.33 毫升丙烯酰胺/双丙烯酰胺 (30/0.8% 瓦特/五), 0.25 毫升1米的 pH 6.8, 0.02 毫升的10% 硫酸铵, 和0.002 毫升的 TEMED。凝胶的尺寸是7.3 厘米 x 8.2 cm x 0.075 cm (l x w x d)。 注意:使用15井梳子的0.75 毫米厚度的最佳灵敏度。</li>
	</ol>
</li>
	<li>将适当数量的6x 载入缓冲液和 dna 样本添加到1.5 毫升的离心管 (例如使用1µL 的6x 加载缓冲区到5µL 的 dna 样本)。</li>
	<li>将凝胶置于垂直电泳室。<ol>
<li>用750毫升的1x 泰缓冲器填充会议厅, 以运行2凝胶。准备1升50x 泰, 使用442克的三基地, 57.1 毫升的冰醋酸, 100 毫升的0.5 米 EDTA pH 8。将蒸馏水加到最后一卷1升。</li>
		<li>将样品装入凝胶中。在100伏的2小时运行凝胶。</li>
	</ol>
</li>
	<li>或者, 用理查德·山姆布鲁克14中发现的配方制备1x 泰琼脂糖凝胶。<ol>
<li>将300毫升的1x 泰加到水平电泳室。运行凝胶45分钟, 在 100 v</li>
	</ol>
</li>
</ol>2. NBT-SG I 染色 (图 1)。
<ol><li>
丙烯酰胺凝胶染色。
	<ol>
<li>准备解决方案: 0.1% 瓦特/v NBT 和 1.2x SG i。 注意:它可以使用溴溴化物, GelRed 和 SYBR 黄金代替 sg i, 但 sg 我有最好的表现。</li>
		<li>将16.75 毫升的 1.2x SYBR 绿色 I 溶液倒入合适大小的容器中。 注意:我们使用吸管尖端盒的盖子作为容器, 并且这些容量被计算用这个容器盖胶凝体;它的尺寸是 11.6 cm x 7.6 cm x 2.6 cm (l x w x d)。</li>
		<li>将凝胶放入溶液中。添加3.25 毫升的0.1% 瓦特/v NBT 溶液给0.2 毫米 NBT 和 1x (相当于2.0 µM) SG I 最终浓度。</li>
		<li>用塑料薄膜封住容器, 用铝箔将容器完全包裹起来, 以阻挡任何环境光线。 注意:该塑料薄膜用于防止飞溅, 避免铝箔与染色液的反应。</li>
		<li>在 60 rpm 的轨道振动筛上摇动25分钟。</li>
		<li>拆卸铝塑盖。暴露在阳光下90分钟最大。检查每5分钟, 直到感兴趣的波段出现。</li>
		<li>要使用蓝光, 准备一个 protoboard 与3发光二极管 (led) 和地方的 led 约5毫米从凝胶表面 (检查染色连续, 因为需要的时间将取决于强度的 LED 光源)。</li>
	</ol>
</li>
	<li>
琼脂糖凝胶染色。
	<ol>
<li>在准备和运行1% 琼脂糖凝胶, 如1.4 节 (尺寸是1厘米 x 6.2 厘米 x 7 厘米; l x w x d), 按照相同的步骤, 在2.1 与这些修改:</li>
		<li>倒入41.9 毫升的 1.2x SG I 溶液进入吸管尖端盒的盖子;它的尺寸是 11.6 cm x 7.6 cm x 2.6 cm (l x w x d)。添加8.1 毫升的 0.1% NBT 溶液。</li>
		<li>用塑料薄膜封住容器, 然后用铝箔将容器完全包裹起来, 以阻挡任何环境光。</li>
		<li>在 60 rpm 的轨道振动筛上晃动1小时。</li>
	</ol>
</li>
</ol>3. 数据分析。
<ol><li>
NBT-SG I 校准曲线。
	<ol>
<li>通过扫描 1200 dpi 获得凝胶图像。</li>
		<li>用于图像分析的开放式软件。使用图像编辑器对图像进行伸直。运行图像密度测量。</li>
		<li>计算曲线下的面积。准备一个信号/信号Max与 DNA 浓度的关系图。</li>
	</ol>
</li>
</ol>4. DNA 恢复。
<ol><li>使用理查德·山姆布鲁克14中发现的配方, 在1x 泰秀中准备1% 琼脂糖凝胶。</li>
	<li>将300毫升的1x 泰加到水平电泳室。在图 2中给出的方案之后, 加载4µL 100 ng/µL pDJ10015质粒和 DNA 阶梯。运行1小时的凝胶 100 v</li>
	<li>马克和重量两2毫升离心管。</li>
	<li>把凝胶切成中间, 以便有两个相同的一半。每一个都应该包含阶梯和样品质粒来比较不同的染色技术。示意图表示形式在图 2中。</li>
	<li>染色一半与 NBT-SG I 如在步骤2。<ol>
<li>用干净的手术刀切出质粒带。将凝胶片保存在一根管子内并称重。</li>
	</ol>
</li>
	<li>染色的其他部分与 sg i 50 毫升的 1x sg i 溶液和缓慢晃动 (60 rpm) 在黑暗中的1小时凝胶。<ol>
<li>将凝胶放在 transilluminator 上, 并暴露2分钟, 用干净的手术刀把质粒带出来。</li>
		<li>将凝胶片保存在另一管中, 称量。 注意:该协议可以暂停在这里, 而保持在-20 ° c 的管。</li>
	</ol>
</li>
	<li>使用商业凝胶萃取试剂盒进行质粒提取。</li>
</ol>5. 质粒正常化。
<ol><li>在1.5 毫升离心管, 添加10µL 提取质粒和60µL I 型水。混合和转移到60µL 石英试管或使用2µL 直接在 Nanodrop。</li>
	<li>测量吸收光谱在230到320毫微米之间。使用以下公式计算样品的质粒浓度: <img alt="Equation 1" src="/files/ftp_upload/56528/56528eq1v3.jpg"> 其中D是应用的稀释因子 (在本例中为 70/10), Ax 是在x nm 上吸收的, 50 是用于 dsDNA 的µg/mL 的转换因子。</li>
	<li>稀释样品至 9.1 ng/µL。</li>
	<li>转换大肠杆菌(大肠杆菌) 有能力的细胞 (如XL10 金) 在井下转换过程16使用5µL 稀释样品。</li>
	<li>计算培养皿上菌落数并注意稀释。使用以下公式计算菌落形成单元 (CFU)17: <img alt="Equation 2" src="/files/ftp_upload/56528/56528eq2v2.jpg"> 其中, D是从初始区域性应用的稀释因子。</li>
	<li>执行不配对 t 检验。</li>
</ol>

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作者没有什么可透露的。

本文介绍了一种灵敏、新颖的基于 NBT 还原的 DNA 染色方法及其在 SG I 中的应用。该协议的关键步骤是凝胶在 NBT-SG I 溶液中的孕育时间和 NBT 的浓度。琼脂糖凝胶的染色时间比丙烯酰胺凝胶更长, 因为琼脂糖凝胶的厚度更大。这种方法在 SDS 存在时不能很好地与 NBT 沉淀物接触。如果凝胶获得紫色的背景, 我们建议准备另一种凝胶, 并减少曝光时间。如果琼脂糖凝胶是染色和没有足够的灵敏度, 建议增...

随着生物化学和分子生物学的进步, 涉及 dna 的研究需要更好的技术来分析 dna。DNA 染色的第一个方法是银染色, 这是非常敏感的, 但缺乏选择性, 不允许样品回收。后来, 荧光 dna 染色的发展使 dna 的选择性定量与样品的回收率的可能性。第一种用于 DNA 定量的荧光染料是溴溴化物1, 它是诱变性的2。然而, 现在有改进的荧光染料, 更安全, 更敏感, 如 GelRed 和 SYBR 绿色 i (SG i)3;但所有这些荧光染料都需要使用紫外线 (UV) transilluminator 或 fluorimeter。
还有其他技术可明显染色的 DNA, 如亚甲基蓝4和晶体紫5,6,7,8,9, 但所有这些都受到降低灵敏度和选择性.唑盐是有机化合物易受减少, 当这发生他们形成一个不溶性和色的甲沉淀10,11。最近, 一些二价唑盐被证明绑定到 DNA 由于其阳性唑环12。
在最近的出版物13中, 提出了一种在聚丙烯酰胺凝胶中染色和量化 DNA 的新的可见技术, 它使用了一种叫做硝基蓝唑 (NBT) 的二价唑盐和荧光染料, 如溴溴化物, GelRed,SYBR 绿我和 SYBR 金。这种反应是在蓝光或阳光的存在下进行的, 与使用紫外线 transilluminator 的 SG I 相比, 可以提高样品的回收率。本文的目的是提供一个详细的协议的染色技术使用唑盐。

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Nitro blue tetrazolium</td>
			<td style="width:227px;">Gold Biotechnology</td>
			<td style="width:307px;">298-83-9</td>
			<td style="width:64px;">reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">SYBR Green I 10000X</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">S7563</td>
			<td>reagent used in the staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Gel extraction kit</td>
			<td style="width:227px;">QIAGEN</td>
			<td style="width:307px;">28704</td>
			<td>used to extract plasmid from the gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">DNA ladder</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">SM 1331</td>
			<td>used to make calibration curves and as reference to cut band in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Agar Agar</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;"></td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">sodium chloride</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1064045000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">yeast extract</td>
			<td style="width:227px;">Becton, Dickinson and Company</td>
			<td style="width:307px;">212750</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">peptone</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">72161000</td>
			<td>used to make LB-ampicillin culture plates&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">TEMED</td>
			<td style="width:227px;">AMRESCO</td>
			<td style="width:307px;">761</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">ammonium persulfate</td>
			<td style="width:227px;">Calbiochem</td>
			<td style="width:307px;">2310</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">acrylamide</td>
			<td style="width:227px;">invitrogen</td>
			<td style="width:307px;">15512-023</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">bisacrylamide</td>
			<td style="width:227px;">SIGMA</td>
			<td style="width:307px;">146072</td>
			<td>used to make polyacrylamide gels</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Tris-base</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1083821000</td>
			<td>used to make TAE buffer and non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">EDTA</td>
			<td style="width:227px;">J.T. Baker</td>
			<td style="width:307px;">8993-01</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Acetic acid</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">1,000,632,500</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">agarose</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">16802</td>
			<td>used to make TAE buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">spectrophotometer</td>
			<td style="width:227px;">Tecan</td>
			<td style="width:307px;">infinite 200 pro</td>
			<td>used to quantify DNA plasmid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">water purificatiom unit</td>
			<td style="width:227px;">Merck</td>
			<td style="width:307px;">Elix 100</td>
			<td>use to make water type 1 (ASTM) used in spectrophotometry and DNA dilutions\</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">distilled water</td>
			<td style="width:227px;"></td>
			<td style="width:307px;">-</td>
			<td>used in gels, culture medium, stainings and general solutions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">HCl</td>
			<td style="width:227px;">J.T. Baker</td>
			<td>9535-03</td>
			<td>used to make non-denaturing polyacrylamide gel tris-HCl buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">6X DNA loading dye</td>
			<td style="width:227px;">Thermo-Fisher</td>
			<td style="width:307px;">R0610</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">5 mm Blue LED</td>
			<td style="width:227px;">Jinyuan electronics</td>
			<td style="width:307px;">SP-021</td>
			<td>light source used in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">protoboard</td>
			<td style="width:227px;">KANDH</td>
			<td style="width:307px;">KH102</td>
			<td>light source support and circuit&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">9 V battery</td>
			<td style="width:227px;">Duracell</td>
			<td style="width:307px;">-</td>
			<td>used to power the LED&#39;s</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:187px;">horizontal electrophoresis chamber</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1704486EDU</td>
			<td>used to make and run agarose gel in integrity assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">vertical electrophoresis kit</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1658002EDU</td>
			<td>used to run polyacrylamide gels in calibration curves</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">power supply</td>
			<td style="width:227px;">Biorad</td>
			<td style="width:307px;">1645050&#160;</td>
			<td>used to power the electrophoresis</td>
		</tr>
	</tbody>
</table>

dna staining method based on formazan precipitation induced by blue light exposure

DNA 染色方法是生物医学研究的重要内容。我们设计了一个简单的方法, 使 DNA 可视化到肉眼的形成一个有色沉淀。它的工作原理是浸泡丙烯酰胺或琼脂糖 DNA 凝胶在 1x (相当于2.0 µM) SYBR 绿色 i (SG i) 和0.20 毫米硝基蓝唑, 产生紫色沉淀的甲时暴露在阳光或特别蓝光。此外, 使用我们的方法染色的琼脂糖凝胶中的氨苄西林抗性质粒进行 DNA 恢复试验。用我们的方法获得的菌落数量比传统的紫外光照射法更大。所描述的方法是快速的, 特异的, 无毒的 DNA 检测, 允许生物分子的可视化, "肉眼" 没有一个 transilluminator, 是廉价和适当的现场使用。由于这些原因, 我们新的 DNA 染色方法对研究和工业都有潜在的好处。

一个紫色的甲沉淀出现在 DNA 的位置 (通过质谱13验证)。在实验中, 加载了一个 DNA 阶梯来制作一个校准曲线 (图 3)。该协议适用于丙烯酰胺和琼脂糖凝胶, 但它的强度较低, 在琼脂糖中需要较长的时间。然而, 使用琼脂糖凝胶允许回收样品使用一个商业可用的试剂盒, 它不干扰的提取。与 SG I 使用 transilluminator (图 4</stron...

Watch this Scientific Journal Video about DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure at JoVE.com

DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

这种方法允许选择性染色和量化的 DNA 在凝胶中浸泡在 SYBR 绿 I/硝基蓝唑溶液, 然后暴露在阳光或蓝光光源。这会产生明显的沉淀, 几乎不需要任何设备, 这使得它非常适合于现场使用。

基于蓝光照射诱发甲沉淀的 DNA 染色方法

DNA staining methods are very important for biomedical research. We designed a simple method that allows DNA visualization to the naked eye by the ...

dna-staining-method-based-on-formazan-precipitation-induced-blue

Research

JoVE Journal

Biochemistry

9.4K 次观看.  Universidad de Chile. 这种方法允许选择性染色和量化的 DNA 在凝胶中浸泡在 SYBR 绿 I/硝基蓝唑溶液, 然后暴露在阳光或蓝光光源。这会产生明显的沉淀, 几乎不需要任何设备, 这使得它非常适合于现场使用。

视频: DNA Staining Method Based on Formazan Precipitation Induced by Blue Light Exposure

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

科研

生物化学

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

通过智能手机实时跟踪DNA片段分离

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

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 ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

电化学检测氘动力学同位素对希瓦氏菌 oneidensis MR-1 细胞外电子传输的影响

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

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 ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

β-淀粉脂质诱导纤维蛋白斑点结构异常的光谱分析

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...