这项工作得到了国家卫生研究院内脏研究司、国家心脏、肺和血液研究所的支持。S.H.得到日本公私合作学生出国留学计划的支持，H.N.和K.Y.获得Valor和V药物海外培训奖学金。

1. SDS-PAGE 并传输到 PVDF 膜
<ol><li>执行 SDS-PAGE，根据相对大小分离蛋白质。 注意:任何商业或自制凝胶系统都没问题。请遵守制造商的协议。</li>
	<li>将分离的蛋白质从凝胶转移到PVDF膜上。 注意:常见的转移方法（半干或湿转移）是合适的。请遵守制造商的协议。</li>
</ol>2. 阻塞PVDF膜
<ol><li>在室温下搅拌时，将膜浸泡在适当的阻塞缓冲区中1小时。 注:根据主要抗体和检测系统，应选择适当的阻塞缓冲区。例如，最典型的阻滞剂是 BSA、脱脂干牛奶、箱蛋白和商用合成聚合物。PBS 和/或 TBS 与 0.1% Tween20 是最常用的缓冲区。此阻塞步骤可在 4 °C 下过夜完成。</li>
</ol>3. 原发性抗体孵化
<ol><li>用蒸馏水准备10%的免疫反应增强剂-1溶液（IRE-1）。漩涡很好。</li>
	<li>用 10% IRE-1 准备稀释的原发抗体。轻轻地和彻底地混合。</li>
	<li>如果使用较大的膜（4 厘米 x 8 厘米 - 8 厘米 x 8 厘米），将 8 mL 的抗体溶液添加到 50 mL 圆锥形离心管中。如果使用较小的膜（2 厘米 x 8 厘米 - 4 厘米 x 8 厘米），将 3 mL 的抗体溶液添加到 14 mL 圆底聚丙烯管（材料表）中。</li>
	<li>用钳子拾取PVDF膜，并短暂地排出阻塞溶液。用戴手套的手指将PVDF膜插入此管中，并确保整个膜粘附在管壁上。当PVDF膜插入管子时，面对最初与凝胶内侧接触的膜的"蛋白质侧"。紧紧地关上帽子。</li>
	<li>将 50 mL 管插入混合烤箱的玻璃瓶中。 注:当 14 mL 管用于此孵化时，将 14 mL 管插入 50 mL 管中，使用一对塑料环架将内管保持在 50 mL 管的中心。</li>
	<li>打开混合烤箱。</li>
	<li>用 6 rpm 旋转孵化膜至少 5 分钟。 注意:确保膜始终粘附在墙壁上，并且（内部）管水平覆盖膜，均匀地覆盖抗体溶液。在实际实验之前校准抗体的稀释和潜伏时间。</li>
</ol>4. 膜洗涤
<ol><li>停止旋转。</li>
	<li>小心地用钳子从管子中取出PVDF膜，并将膜放入一个装有50 mL PBS-T的容器中。</li>
	<li>用 PBS-T 短暂地冲洗膜。</li>
	<li>用蒸馏水冲洗容器中的膜，直到没有气泡出现。这是为了去除大部分抗体。</li>
	<li>将PVDF膜从容器转移到含有250 mL PBS-T的沙拉微调器。</li>
	<li>将过滤器篮放入微调器中。确保盖子安全放置。</li>
	<li>激活微调器并运行大约 20-30 s。</li>
	<li>丢弃溶液，用蒸馏水短暂冲洗微调器内部。</li>
	<li>再次将 250 mL 的 PBS-T 添加到微调器中。</li>
	<li>重复步骤 4.6 和 4.7。</li>
</ol>5. 与二次抗体一起孵育
<ol><li>用蒸馏水准备10%的免疫反应增强剂-2溶液（IRE-2）。漩涡很好。</li>
	<li>用 10% IRE-2 准备稀释的次要抗体。轻轻地和彻底地混合。</li>
	<li>选择第 3.3 步中注述的抗体溶液和管的体积。</li>
	<li>用钳子拾取PVDF膜，并短暂地排干溶液。将 PVDF 膜插入第 3.4 步所述的管子中。</li>
	<li>将 50 mL 管插入混合烤箱的玻璃瓶中。</li>
	<li>打开混合烤箱。</li>
	<li>用 6 rpm 旋转孵化膜至少 5 分钟。确保膜始终粘附在墙壁上，并且（内部）管水平，以便用抗体溶液均匀地覆盖膜。 注:在实际实验之前校准孵化时间。当次要抗体被荧光结合时，在黑暗中进行孵化。</li>
</ol>6. 膜洗涤
<ol><li>按照步骤 4.1 到 4.10。</li>
</ol>7. 图像采集
<ol><li>化学发光检测<ol>
<li>将一块半移植柔性薄膜（10厘米×15厘米）放在平坦的表面上。</li>
		<li>在 5 mL 管中混合化疗基板的 2 个组件。</li>
		<li>立即将混合基板的1.5 mL转移到半移植柔性薄膜上，并将PVDF膜放在它身上。然后，将基板溶液的其余部分转移到膜上。</li>
		<li>在半透明柔性薄膜上用混合基板孵化PVDF膜1分钟。</li>
		<li>用钳子拾取PVDF膜，并短暂地排出基板溶液。</li>
		<li>将膜夹在一对透明胶片之间。</li>
		<li>在化学发光模式下获取图像。</li>
	</ol>
</li>
	<li>荧光检测<ol>
<li>用钳子拾取PVDF膜，并短暂地排干溶液。</li>
		<li>将膜夹在一对透明胶片之间。</li>
		<li>在荧光模式下获取图像。 注:步骤 7.1 和 7.2 应在成像机附近执行，以确保最大灵敏度。</li>
		<li>当背景信号高时，在装有 80-100 mL PBS-T 的容器中执行洗涤步骤:10 分钟冲洗 3 次用于原发抗体，5 分钟冲洗 6 次用于次要抗体。 注:用10%IRE溶液稀释的原辅抗体可使用8-10次，而不会失去灵敏度6。解决方案应保持在4°C长达1个月。</li>
	</ol>
</li>
</ol>

<ol>
	<li>MacPhee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+improving+Western+blot+analysis.">Methodological considerations for improving Western blot analysis.</a> Journal of Pharmacol Toxicology Methods. 61 (2), 171-177 (2010).</li><li>Janes, K. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Western+blotting":+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">"Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112 (2), 195-203 (1981).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Ciaccio, M. F., Wagner, J. P., Chuu, C. P., Lauffenburger, D. A., Jones, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, 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=A+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. 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作者已宣布没有与本文内容直接相关的相互竞争的利益。

西方污点是一种广泛使用的分析技术，用于检测40年前7、8年前开发的特定蛋白质。此后，该技术不断发展，随后的创新提高了技术2、9、10、11、12、13的灵敏度、速度和定量。此处介绍的增强型超高速西?...

西方的污点（也称为免疫虫）是一种强大而基本的技术，跨越广泛的科学和临床学科。该技术用于检查蛋白质1的存在、相对丰度、相对分子质量和转化后修饰。结合数字图像分析，该方法可以可靠地分析蛋白质的丰度和蛋白质的修饰2。虽然西方的污点是例行公事，但它是一种耗时和劳动密集型的方法。需要用膜长期培养抗体。在这里，我们描述了对孵化方法的修改，该方法克服了这一限制，而不会牺牲灵敏度。
在膜的孵化过程中，抗体漂浮在溶液中，而抗原则固定在膜上。由于其亲和力高，抗体与抗原结合的速度比抗体从散装溶液扩散到膜的速度要快。这会产生低浓度的"消耗层"（图 1）。较远的抗体可能需要数小时才能通过被动扩散到达膜，而被动扩散是导致该技术长期潜伏的主要因素。此效果称为大众运输限制 （MTL）3。有人提出，重复排空和补充含抗体溶液可能会破坏耗竭层，克服MTL4。在这里，我们设计了一种独特的技术，实现这种循环排水和补充 （CDR） 概念，以减少 MTL 在传统的西方印迹协议中的影响，并显著缩短所需的潜伏期。
为了在短的潜伏时间内保持检测灵敏度，我们采用了提高免疫力增强技术，加速抗原抗体反应，从而提高了信号噪声比5。许多市售的免疫增强剂 （IRE） 由 2 个组件组成（解决方案 1 和 2，见 材料表）。IRE的专有成分或作用机制尚未披露，但我们先前发现，IRE在固体相绑定检测中减少了抗原和抗体之间的分离常数，表明亲和力的增强至少部分地对IRE6的增强作用负有责任。CDR 与 IRE 的结合产生了一种超高速的西方印迹处理方案，可在不牺牲灵敏度的情况下缩短整个过程时间。

<table>
	<tbody>
		<tr>
			<td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td>
			<td>Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Apollo Transparency Film for Laser Printers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any kinds of Laser Printer Transparency Film are fine.</td>
		</tr>
		<tr>
			<td>Azure Imaging System 600</td>
			<td>Azure Biosystems</td>
			<td>Azure 600</td>
			<td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td>
		</tr>
		<tr>
			<td>Can Get Signal Immunoreaction Enhancer Solution</td>
			<td>TOYOBO</td>
			<td>NKB-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CARNATION Instant Nonfat Dry Milk</td>
			<td>Carnation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from sheep</td>
			<td>GE Healthcare</td>
			<td>NA9310V</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from donkey</td>
			<td>GE Healthcare</td>
			<td>NA9340V</td>
			<td></td>
		</tr>
		<tr>
			<td>HYBAID Micro-4</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF Membrane, 0.45 &#181;m pore size</td>
			<td>Millipore Sigma</td>
			<td>IPVH304F0</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-32211</td>
			<td></td>
		</tr>
		<tr>
			<td>KPL LumiGLO Reserve Chemiluminescent Substrate</td>
			<td>seracare</td>
			<td>5430-0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#223; actin antibody</td>
			<td>Millipore Sigma</td>
			<td>A5316</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (PBS)</td>
			<td>LICOR</td>
			<td>927-40100</td>
			<td>Blocking Buffer for fluorescent detection</td>
		</tr>
		<tr>
			<td>OXO Salad Spinner</td>
			<td>OXO</td>
			<td>32480V2B</td>
			<td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td>
		</tr>
		<tr>
			<td>Parafilm Sealing Film</td>
			<td>Bemis</td>
			<td>Parafilm M PM996</td>
			<td>semi-transparent flexible film</td>
		</tr>
		<tr>
			<td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Rings to hold 14 ml tube in the center of 50 ml tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared in a machine shop</td>
		</tr>
		<tr>
			<td>Rabbit anti-6X His tag antibody</td>
			<td>Abcam</td>
			<td>ab9108</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Millipore Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultra-high-speed western blot using immunoreaction enhancing technology

西方的污点（也称为免疫虫）是生物医学研究的规范方法。它通常用于确定特定蛋白质的相对大小和丰度以及转化后蛋白质的修饰。这种技术具有丰富的历史，由于其简单性，仍然被广泛使用。然而，西方的印迹程序著名的需要几个小时，甚至几天，完成，一个关键的瓶颈是长期的潜伏时间，限制其吞吐量。由于抗体从散装溶液缓慢扩散到膜上的固定抗原，因此需要这些培养步骤:膜附近的抗体浓度远远低于散装浓度。在这里，我们提出了一项创新，通过循环排空和补充抗体溶液 （CDR） 来改善抗原结合，从而显著缩短这些潜伏间隔。我们还利用了增强免疫力的技术来保持检测的灵敏度。CDR方法与商业免疫增强剂相结合，提高了输出信号，大大缩短了抗体潜伏时间。由此产生的超高速西部污点可以在20分钟内完成，而不会失去任何灵敏度。该方法可同时使用化学发光和荧光检测方法应用于西方污点。这种简单的协议使研究人员能够更好地探索许多样本中蛋白质表达的分析。

这个例子说明了CDR方法以及免疫增强技术对西方污点的有效性。静态孵化是在半透明的柔性薄膜上进行的，其中PVDF膜与抗体溶液一起孵育，而CDR孵化则通过含有膜和抗体溶液（电影）的旋转管在混合烤箱中。 图2 显示的抗原和抗体数量分别为西斑化学发光检测所必需的。在静态和 CDR 孵化中，使用 IRE 溶液提高了灵敏度:IRE 将检测 é actin 所需的最低细胞溶解物 （抗原） 数量...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

通过循环排泄和补充 （CDR） 技术与免疫增异剂一起改进抗原抗体结合的动能，开发出一种超高速西方印迹技术。

使用免疫增强技术的超高速西布洛特

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Universidad de Cartagena UDEC

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JoVE Journal

Biochemistry

5.8K 次观看.  National Institutes of Health. 通过循环排泄和补充 （CDR） 技术与免疫增异剂一起改进抗原抗体结合的动能，开发出一种超高速西方印迹技术。

视频: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

高通量, 用定量斑点杂交分析法绝对测定组织层中选定蛋白质的含量 (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

Studying Protein Import into Chloroplasts Using Protoplasts

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many secondary metabolites and lipids. Chloroplasts require a large number of proteins for these various physiological processes. Over 95% of chloroplast proteins are nucleus-encoded and imported into chloroplasts from the cytosol after translation on cytosolic ribosomes. Thus, the proper import or targeting of these nucleus-encoded chloroplast proteins to chloroplasts is essential for the proper functioning of chloroplasts as well as the plant cell. Nucleus-encoded chloroplast proteins contain signal sequences for specific targeting to chloroplasts. Molecular machinery localized to the chloroplast or cytosol recognize these signals and carry out the import process. To investigate the mechanisms of protein import or targeting to chloroplasts in vivo, we developed a rapid, efficient protoplast-based method to analyze protein import into chloroplasts of Arabidopsis. In this method, we use protoplasts isolated from leaf tissues of Arabidopsis. Here, we provide a detailed protocol for using protoplasts to investigate the mechanism by which proteins are imported into chloroplasts.

Here we describe a protocol to express proteins into protoplasts by using PEG-mediated transformation method. The method provides easy expression of proteins of interest, and efficient investigation of protein localization and the import process for various experimental conditions in vivo.

原生质体在叶绿体中导入蛋白质的研究

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

科蒂醇测量在考拉 （菲斯科拉托斯西尼鲁斯） 毛皮

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally identified by harnessing the competitions between amino acids and their analogs. However, this analog-based method is of low accuracy, and proper analogs for specific amino acids are limited. Here, we present an alternative strategy that enables an accurate, sensitive, and high-throughput screening of amino acid overproducers using rare-codon-rich markers. This strategy is inspired by the phenomenon of codon usage bias in protein translation, for which codons are categorized into common or rare ones based on their frequencies of occurrence in the coding DNA. The translation of rare codons depends on their corresponding rare transfer RNAs (tRNAs), which cannot be fully charged by the cognate amino acids under starvation. Theoretically, the rare tRNAs can be charged if there is a surplus of the amino acids after charging the synonymous common isoacceptors. Therefore, retarded translations caused by rare codons could be restored by feeding or intracellular overproductions of the corresponding amino acids. Under this assumption, a selection or screening system for identifying amino acid overproducers is established by replacing the common codons of the targeted amino acids with their synonymous rare alternatives in the antibiotic resistance genes or the genes encoding fluorescent or chromogenic proteins. We show that the protein expressions can be greatly hindered by the incorporation of rare codons and that the levels of proteins correlate positively with the amino acid concentrations. Using this system, overproducers of multiple amino acids can be readily screened out from mutation libraries. This rare-codon-based strategy only requires a single modified gene, and the host is less likely to escape the selection than in other methods. It offers an alternative approach for obtaining amino acid overproducers.

This study presents an alternative strategy to the conventional toxic analog-based method in identifying amino acid overproducers by using rare-codon-rich markers to achieve accuracy, sensitivity, and high-throughput simultaneously.

使用稀有的科顿富标记识别氨基酸过度生产者

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

This protocol presents a technique for probing protein-protein interactions using glutathione-linked donor beads with GST-fused TPR-motif co-chaperones and acceptor beads coupled with an Hsp90-derived peptide. We have used this technique to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

使用同质珠为基础的检测的查佩龙-科查佩龙相互作用研究

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...