这项工作是由来自美国国立卫生和乔治E ·休伊特基金会奖学金（美联社）研究院的拨款支持。

凝胶电泳 技术说明 ：在启动程序之前，您的蛋白质样品准备。 在这第一步，样品中的蛋白质，根据其分子量使用变性聚丙烯酰胺凝胶电泳（PAGE）分离。锂十二烷基硫酸钠（LDS）中加载NuPAGE ® LDS样品缓冲液保持在变性状态，多肽，蛋白质样品已在70 ° C加热10分钟一次。强还原剂配合使用，以消除二级和三级结构（数码地面电视，打破二硫键）。此外，样品的蛋白质成为带负电荷的摩门教覆盖，并因此带正电的电极通过丙烯酰胺凝胶网状走向。根据分子量（公斤道尔顿，KDA测量），这使得他们的分离。 的Invitrogen公司网站2上可以找到详细的一步一步的样品制备和网页程序的协议，或在NuPAGE 技术指导3。 技术秘诀<ol><li> Invitrogen公司NuPAGE双三不连续缓冲系统，电泳迁移率的蛋白质和随后的凝胶分离范围取决于两个因素：一）acrylamid凝胶浓度（与更大的丙烯酰胺的浓度，导致较低的分辨率更好分子量的蛋白质）和ii）运行缓冲液，公开资讯观测站或MES尾随离子。要选择适当的组合，你想实现的分离范围，是指凝胶迁移图1。凝胶的厚度和井数将取决于您计划分别加载，样品的数量和数量。 </li><li>当你加载到您的样品的凝胶井，至少有一条车道被保留为分子量标记（或梯形）。这些蛋白质标准市售的几家公司，通常由一个有定义的分子量染色蛋白质的混合​​物，使之形成可见光波段，使您能够按照移民进度。选择一个兼容凝胶决议的分子量范围。 </li><li>为了实现好，甚至迁移模式，我们建议您凝胶井装上一个类似的样品或1X LDS样品缓冲量。 </li></ol>转让 技术说明 ：转让步骤开始之前，准备转移的缓冲液（用10％甲醇1X）和预冷却到4 ° C的寒冷的房间。 为了使抗体检测的蛋白质，他们转移到硝酸纤维素膜，凝胶electroblotting。基于疏水相互作用，以及负责膜和蛋白质之间的相互作用蛋白结合。 （聚偏二氟乙烯（PVDF）膜也可以被用来作为一种替代，在这种情况下，聚偏氟乙烯膜需要在甲醇预湿，使用前至少30秒。） 一个详细的一步一步的协议的传输过程中可以发现在参考文献4，如果您使用Bio - Rad公司的小型跨印迹电泳转移细胞，或引用2-3，如果您的实验室是配备与Invitrogen公司的XCell II™吸干模块。 作为这一过程的结果，暴露在一个薄的表面层的蛋白质，并准备进行检测。可以检查可逆丽春红染料膜染色的蛋白质从凝胶转移到膜的均匀性和整体效益。 丽春红S染色程序（可选）： <ol><li>转移的蛋白质后，放置在孵化盘膜（蛋白质朝上）。 </li><li>加入足够的丽春红S染色覆盖膜，轻轻摇动孵育至少30秒。 </li><li>用蒸馏水冲洗膜，直到背景清晰。 </li><li> Destain运行2-3分钟，蒸馏水膜。 </li><li>将膜在封闭液（见下文）。 </li></ol> 技术提示： <ol><li>我们建议你的膜蛋白转移前的铅笔标签，以表示在其中的蛋白质被暴露的侧面和您的样品的方向。 </li><li>丽春红S染色和分子量标记可以用来探测不同的抗体产生的部分削减后的转印膜</li></ol>免疫检测： 技术说明 ：这种免疫检测过程只作为指引。优化可能需要为每个蚂蚁ibody;具体的信息可以发现在大部分市售的抗体数据表（例如，工作液，培养时间等）。 在这最后的过程中，目标蛋白质将使用特定抗体检测，并会出现作为一个乐队在胶片上。乐队中的位置是依赖靶蛋白的分子量，而带的强度取决于靶蛋白目前量上。 通常，这是后三个子步骤实现： <ol><li>阻塞：为了避免与非特异性抗体的相互作用的膜（膜的多余的空间是用一个通用的蛋白质的稀溶液覆盖）。 </li><li>探讨：感兴趣的蛋白质是由一个特定的<a title="主要抗体" href="http://en.wikipedia.org/wiki/Primary_antibody">初级抗体</a>检测。冲走后未结合的抗体，膜接触到不同的抗体与记者<a title="酶" href="http://en.wikipedia.org/wiki/Enzyme">酶</a> ， <a title="辣根过氧化物酶" href="http://en.wikipedia.org/wiki/Horseradish_peroxidase">辣根过氧化物酶</a> （HRP）。这个二级抗体是针对物种的特定部分抗体（ 如抗兔二抗将绑定到任何兔源抗体）。 </li><li>检测：化学发光剂作为基质，将暴露时，发光二级抗体的HRP。这种反应产生发光的地方，在探测蛋白质的量成比例。然后检测到的光感光胶片。 </li></ol>下面提供一个通用的一步一步的程序。其他详细的程序中可以找到的ECL Plus西方印迹检测试剂说明书5，或在最所附数据表市售的抗体。孵化时间，抗体的稀释，和封锁和清洗解决方案，每个抗体凭经验优化。 免疫检测程序： <ol><li>阻止非特异性结合，培育足够的体积PBS 1％酪蛋白阻塞的解决方案，以覆盖整个膜。放在摇杆在室温下至少30分钟或4℃过夜° C。 </li><li>孵育与您的主要的抗体（蛋白质利益的具体）的膜。应在封闭液稀释的主要抗体。对于大多数的抗体，实现完整的具有约束力的潜伏期1-2个小时后在室温下轻轻摇动下。此外，孵化步骤可以进行4℃过夜° C至增加信噪比。 </li><li>删除的解决方案（丢弃或保持在4 ° C至-20 ° C的重复使用），快速洗膜一次。然后用PBS，0.05％吐温20（PBST）在室温下剧烈摇晃3X10分钟。 </li><li>孵育膜与HRP耦合二次封闭液稀释的抗体。选择抗体识别的提出的主要抗体是其中的物种的IgG部分。潜伏期是在室温下轻轻摇动的温度为1小时。 </li><li>丢弃的解决方案和快速洗膜一次。然后在室温下剧烈摇晃PBST 3X10分钟。 </li><li>根据制造商的指令，继续进行化学发光检测。我们使用从通用电气医疗集团（具体说明见参考文献5）的ECL Plus Western印迹检测试剂。 </li><li>请将您的膜保鲜膜，或邮袋和内的放射自显影盒。确保排出多余的液体，并删除所有气泡。 </li><li>使感光胶片膜在黑暗的房间。开始1分钟的曝光时间，并根据所需的信号强度进行调整。 </li></ol> 技术提示： <ol><li>阻断非特异性结合，是通过放置在稀溶液中的蛋白质膜。我们通常使用1％的酪蛋白， <a title="牛血清白蛋白" href="http://en.wikipedia.org/wiki/Bovine_serum_albumin">但牛血清</a>白蛋白（〜2％BSA）或不脱脂奶粉（〜5％），可以作为替代使用。 </li><li>可用于整个过程，而不是PBS的Tris缓冲液（TBS）。我们推荐使用的TBS如果您计划您的膜探针与磷酸抗体。 </li><li>利用辉光在黑暗的贴纸（如Stratagene的Glogos ® II Autorad标记）的放射自显影盒的底部挖掘，将帮助您对齐在分析步骤中，您的电影和你的膜。 </li></ol>分析现在你已经暴露了你的电影，你会发现，在实践中，并非所有的西部片揭示蛋白质作为一个漂亮的单波段。 AdditionaL波段也可能出现由于小学和中学的抗体非特异性结合。优化免疫检测程序，可以减少这种背景信号。此外，适当的控制（例如，未转染细胞，siRNA处理的细胞等）将是有益的，以确定您的抗体的特异性和靶蛋白膜的确切位置。 胶片上的标记后染蛋白膜的标准带的位置，画出每个分子量的日志 对他们的相对移动（X轴）（y轴）的蛋白质标准。相对流动性（RF）的蛋白质，从它的起源点（凝胶的顶部）的相对距离跟踪染料或低分子量标记移动的距离比使用的术语，提出（凝胶前） 。确定的标准曲线，获得斜率和截距值的回归线。未知靶蛋白（分子量大小），估计使用其Rf和修改了以下方程： 日志分子量=（斜率）（靶蛋白的流动性或RF）+ Y轴截距 （详细说明见参考文献6）。 通过比较靶蛋白的峰强度的结构蛋白（如微管蛋白或肌动蛋白）或如GAPDH看家基因产品的表达水平近似。这种所谓的“加载控制”，不应该改变和样品之间透露使用特定的抗体。图像可能会进一步通过密度测量，以评估蛋白表达的相对量，光密度的量化结果进行分析。

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+Migration+chart:+.">Gel Migration chart: .</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+western-blot+protocol+from+the+Invitrogen+website.">Complete western-blot protocol from the Invitrogen website.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NuPAGE+technical+guide.">NuPAGE technical guide.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+Manual,+Mini+Trans-Blot+Cell.">Instruction Manual, Mini Trans-Blot Cell.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECL+Plus+Western+blotting+detection+reagents+instruction.">ECL Plus Western blotting detection reagents instruction.</a> , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+protein+molecular+weights+by+SDS+gel+electrophoresis,+GE+Healthcare+Website.">Estimation of protein molecular weights by SDS gel electrophoresis, GE Healthcare Website.</a> , .</li><li>Yeromin, A. V., 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=Molecular+identification+of+the+CRAC+channel+by+altered+ion+selectivity+in+a+mutant+of+Orai.">Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.</a> Nature. 443, 226-229 (2006).</li><li>Zhang, S. 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=Store-dependent+and+-independent+modes+regulating+CRAC+channel+activity+of+human+Orai1+and+Orai3.">Store-dependent and -independent modes regulating CRAC channel activity of human Orai1 and Orai3.</a> J. Biol. Chem. 283, 17662-17671 (2008).</li><li>Penna, , 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+CRAC+channel+consists+of+a+tetramer+formed+by+Stim-induced+dimerization+of+Orai+dimers.">The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.</a> Nature. , .</li></ol>

这里介绍的程序使用双三凝胶与MES作为使用Invitrogen公司NuPAGE NOVEX电泳系统变性PAGE的例子运行缓冲区。此外，这个预制凝胶系统允许下变性或非变性条件以及蛋白质分离，容纳了广泛的分子量范围（1-200 kDa的双三凝胶为36-400 kDa的三醋酸凝胶）。根据你的实验，你可以选择只遵循此处描述的Western印迹技术的一部分，使用直接凝胶染色程序（例如，银或考马斯染色）或其他免疫检测方法（例如，比色法或荧光）。 </...

western blotting using the invitrogen nupage novex bis tris minigels

免疫印迹（或免疫印迹）是一个标准的实验室程序，调查核实的一种蛋白质的表达，确定不同样品中的蛋白质目前的相对量，和免疫共沉淀实验分析结果。在此方法中，检测到目标蛋白质与特定抗体在组织匀浆或提取样品。根据分子量的蛋白质的分离是通过使用变性的SDS - PAGE。转移到膜后，靶蛋白的探测与特定的抗体，通过化学发光法检测。 自第一次描述，Western印迹技术已发生了一些改进，包括预制凝胶和用户友好的设备。在我们的实验室中，我们选择使用自Invitrogen市售NuPAGE电泳系统。这是一个创新的中性pH值，不连续的SDS - PAGE，预制小凝胶体系。该系统提出了一个较长的保质期从8个月至1年不等的预制凝胶ii）一个广泛的分离范围从1到400 kDa的分子量不同的类型，包括传统Laemmli技术的几个优势：一）凝胶使用;及iii）更大的灵活性（丙烯酰胺的比例，凝胶型，和运行缓冲液的离子组成范围）。 在这个视频文章中描述的程序利用了4-12％，二，三梯度凝胶和MES运行说明如何使用Invitrogen公司NuPAGE电泳系统进行免疫印迹的缓冲区，双三不连续缓冲系统。在我们的实验室中，我们已取得了良好的和可重复的结果，采用Western印迹方法的各种生化应用。

Watch this Scientific Journal Video about Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels at JoVE.com

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

这篇技术文章介绍了一个标准的程序，使用市售NuPAGE电泳迷你凝胶体系自Invitrogen Western印迹。

免疫印迹使用Invitrogen公司NuPage NOVEX双三MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

western-blotting-using-the-invitrogen-nupage-novex-bis-tris-minigels

Research

JoVE Journal

Biology

36.9K 次观看.  University of California, Irvine (UCI). 这篇技术文章介绍了一个标准的程序，使用市售NuPAGE电泳迷你凝胶体系自Invitrogen Western印迹。

视频: Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

内淋巴管闭塞手术诱导的内淋巴积水

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

科研

生物学

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

免疫印迹检测样品制备

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Solid Phase Synthesis of a Functionalized Bis-Peptide Using &quot;Safety Catch&quot; Methodology

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

一个官能二肽的固相合成使用“安全锁扣”方法论

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

使用高解析度电脑断层可视化的三维结构和功能的植物血管

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

V3 无污渍工作流，适用于西式印迹中实用、方便和可靠的总蛋白质加载控制

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

最快在西镇：对经典蛋白质印迹分析现代风味

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...