Name: a guide to modern quantitative fluorescent western blotting with troubleshooting strategies
Uploaded: 2014-11-20T16:00:00.000+00:00
Duration: 11 min 1 s
Description: Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

我们想感谢下列人员或资金支持:BBSRC研究所战略方案基金 - CF＆TMW; BBSRC东生物DTP资金 - LG;爱丁堡的达尔文信托 - MLH。我们还要感谢巴里·麦科尔医生的许可，包括在这个手稿的TREM2优化。

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A., Foley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+signaling+events+in+Drosophila+S2+cells.">Quantitative evaluation of signaling events in Drosophila S2 cells.</a> Biol Proceed Online. 10 (1), 20-28 (2008).</li><li>Wishart, T. M., 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=Differential+proteomics+analysis+of+synaptic+proteins+identifies+potential+cellular+targets+and+protein+mediators+of+synaptic+neuroprotection+conferred+by+the+slow+Wallerian+degeneration+(Wlds)+gene.">Differential proteomics analysis of synaptic proteins identifies potential cellular targets and protein mediators of synaptic neuroprotection conferred by the slow Wallerian degeneration (Wlds) gene.</a> Mol Cell Proteomics. 6, 1318-1330 (2007).</li><li>Silva, J. M., McMahon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fastest+western+in+town:+a+contemporary+twist+on+the+classic+western+blot+analysis.">The fastest western in town: a contemporary twist on the classic western blot analysis.</a> J. Vis. Exp. (84), 51149 (2014).</li></ol>

The authors have nothing to disclose.

由于考虑和计划是之前的任何实验必不可少，并且可以最终确定所使用的技术的成功。用WB可以呈现过多的潜在绊脚石试图选择合适的抗体，转移和可视化方法使用时，在蛋白质检测的进步。幸运的是，通过仔细的检查表和QFWB可常规用于确定蛋白质的存在和样品之间日益微妙的表达差异适当的控制措施。该协议提供了一个全面的指南，以荧光定量免疫印迹以及一些解决问题的策略，以避免和/或克服一些与此相关的许多常见的陷阱。 用来维持灵敏度，将获得的关键步骤真正量化和可比较的测量包括:1）鲁棒蛋白提取自组织样品; 2）样品制备; 3）准确的蛋白质插件中克以总蛋白质分析测定; 4）优化利用I-Blot检测蛋白的转移; 5）使用红外成像器和相关联的软件准备一级和二级抗体在阻断含0.1％吐温20缓冲液，和6）正确的可视化和分析。 红外荧光检测是真正的量化，并与更传统的电化学发光检测技术3,11相比提供了更高的灵敏度。该检测系统是多面性的，因此并不限定于QFWB。该系统能够免疫组化标记成像在低功耗，允许可视化的整个组织切片12和量化。这是未来发展潜力的一个区域在分辨率方面可能远远看见红色的成像媲美常规免疫捕获与传统的显微镜定量评估方面。 然而，与在P微妙的变化更加敏感rotein表达关键的是要确保变性保持在最低限度和控制措施是严格的具有强大的协议。这开始于严谨蛋白提取组织样本随后生产总蛋白质染色的凝胶以提供保证样品装载是均匀的，第一和第二抗体，以确定是否检测是真实的，对于测试制造商的指导方针来传输优化次，以获得有效的蛋白转移。 然而，即使当用于白平衡的条件进行了优化，但仍然可以使用运行西部片可能未得到充分这里探讨了相关的问题。这些包括但不限于因素，包括蛋白质溶解和选择的提取缓冲液。某些缓冲剂能与蛋白质浓度测定干扰，和一些组织中是特别难以溶解，因此需要更强大的技术，例如利用自动macera鼎密封容器如M管连同Mac机离解。此外，对于存储提取和非萃取材料在-80℃下的简单的控制措施可以是提取后立即获得最佳标记和具有较差的结果周后之间的差。 现代QFWB方法被证明是用于捕获在蛋白质表达的细微差别更敏感，更通用的允许同时双标记3时相比，如ECL旧的技术。至关重要的是，免疫​​印迹协议是强大和易于重复进行准确的定量和统计分析。这个协议是敏感的和强大到足以被常规用于检测蛋白的跨各种不同的组织样本和物种3，并且允许低和高丰度蛋白质的定量相同QFWB内，因此减少了耗材使用以及每个实验1时间1此外，该技术的敏感性增加使得越来越流行的验证- 组学研究9,14然而精度是至关重要的，并包含合适的控制措施，必须遵守从而避免错误的数据采集。

Western印迹法（WB）是最初在20世纪70年代后期，以确定所关注的蛋白的存在或不存在下在一个复杂的生物样品，如组织匀浆1的分析技术。通常被称为该蛋白质的免疫印迹中，由于密钥抗体 - 抗原相互作用，该方法包括5个不同的步骤:蛋白质通过等电点为1）电泳分离; 2）转移到硝酸纤维素或聚偏二氟乙烯（PVDF）膜; 3）使用特异于目的蛋白质的初级抗体标记; 4）培养用针对第一抗体的第二抗体;和5）的可视化。 可视化的方法已演变随着时间的推移，以提高安全性和灵敏度。一些第一转债的进行了使用放射性标记的标签，然后进展到比色，然后更广泛使用的化学发光（ECL）方法。 Radioactivi一节被直接标记到探针的特异性抗原，而比色和ECL方法使用用酶记者间接标记技术，如碱性磷酸酶或链霉抗辣根过氧化物酶（HRP）2。的发色或发光产物的标记的强度是使用光密度测定，由此，信号强度，或强或弱，表明样品中目的蛋白质的或多或少的存在下进行测定。 ECL是比较敏感的，所以青睐的方法2，但所有的3种方法最初开发于X射线胶片与随后成立3更先进的数码影像技术。 WB的数字成像的进步不仅允许研究者来确定感兴趣的蛋白的存在或不存在的，但也允许一个推理到一个选定的蛋白质的表达水平，当与其他样品相比，因此可被称为"半定量＆＃8221 ;.最近，一个真正的定量和更灵敏的免疫印迹技术已经发展，由此进行荧光测定的水平，直接关系到一个样品中的量和单个蛋白的表达:定量的荧光蛋白质印迹（QFWB）。 当与ECL标记比较QFWB，利用荧光第二抗体的产生线性检测曲线4。这是相对于电致化学发光技术，其中，信号的线性度通常发生在低于5微克和信号饱和度低的蛋白质负载直接相关的蛋白的表达， 即，与遍在表达的持家基因5,6。这种差异很可能是由一个更大数量的结合位点，可用于抗生物素蛋白ECL底物结合到生物素化的次级，导致潜在的信号饱和的较高可能性引起的。这是被称为外核层的ECL基础免疫的重要原因之一Y"半定量"7。测定的表达水平的细微差异时，可能导致不准确的测量信号的饱和点是至关重要的。近年来广泛蛋白质组技术细节不断增加的敏感性和鉴别的微妙表达差异的出现，导致了日益增加的依赖真正的定量免疫印迹法进行验证实验，8,9。敏感，稳健​​和真正的定量方法的应用是至关重要的。 很多"优化"免疫印迹方法已经使用了独立的实验室，这经常被证明难以成立或复制，由于可能无法在正式的文件协议明显微妙的方法上的调整。这是一个既定的和强大的协议QFWB并另外提供了宝贵的战略，解决常见问题THA实现在t可能出现。 该协议最初是为与鼠脑匀浆进行了优化，但已经被在广泛的组织样本和物种4,9,10的有效利用。需要具体问题进行故障排除潜在协议的变化都包括在内。

<table><tbody><tr><td>RIPA Buffer</td><td>Fisher Scientific UK Ltd</td><td>10230544</td><td></td></tr><tr><td>M Tubes</td><td>Miltenyi Biotec Inc.</td><td>130-093-236</td><td></td></tr><tr><td>iBlot Transfer Stack, PVDF Regular</td><td>Life Technologies, UK</td><td>IB401001</td><td></td></tr><tr><td>MagicMark XP Western Protein Standard (20-220 kDa)</td><td>Life Technologies, UK</td><td>LC5602</td><td>Use in gel 1 for Western blotting</td></tr><tr><td>SeeBlue Pre-stained protein standard</td><td>Life Technologies, UK</td><td>LC5625&amp;nbsp;</td><td>Use in gel 2 Total protein stained gel</td></tr><tr><td>NuPAge LDS Sample buffer 4x</td><td>Life Technologies, UK</td><td>NP0007</td><td></td></tr><tr><td>NuPAGE MES SDS Running Buffer (for Bis-Tris Gels only) (20x)</td><td>Life Technologies, UK</td><td>NP0002</td><td></td></tr><tr><td>NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well</td><td>Life Technologies, UK</td><td>NP0322BOX</td><td></td></tr><tr><td>Phosphate buffered saline tablet</td><td>Sigma-Aldrich, UK</td><td>P4417-100TAB</td><td></td></tr><tr><td>Micro BCA, Protein Assay Kit</td><td>Fisher Scientific UK Ltd</td><td>10249133</td><td></td></tr><tr><td>Odyssey blocking buffer</td><td>Li-Cor Biosciences</td><td>P/N 927-40000</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Rabbit IgG (H+L), 0.5 mg</td><td>Li-Cor Biosciences</td><td>926-68071</td><td></td></tr><tr><td>IRDye 680RD Donkey anti-Mouse IgG (H+L), 0.5 mg&amp;nbsp;</td><td>Li-Cor Biosciences</td><td>926-68072</td><td></td></tr><tr><td>IRDye 800CW Goat anti-Rabbit IgG (H + L), 0.5 mg</td><td>Li-Cor Biosciences</td><td>926-32211</td><td></td></tr><tr><td>IRDye 800CW Donkey anti-Goat IgG (H + L), 0.5 mg</td><td>Li-Cor Biosciences</td><td>926-32214</td><td></td></tr><tr><td>ODYSSEY CL Infra-red imager</td><td>Li-Cor Biosciences</td><td></td><td>Call for quotation</td></tr><tr><td>iBlot 7-Minute Blotting System</td><td>Life technologies, UK</td><td></td><td>This model is no longer in production</td></tr><tr><td>InstantBlue Protein stain</td><td>Expedeon, UK</td><td>ISB1L</td><td></td></tr><tr><td>Revitablot western blot stripping buffer&amp;nbsp;</td><td>Rockland Immunochemicals Inc.</td><td>MB-085-0050</td><td></td></tr></tbody></table>

a guide to modern quantitative fluorescent western blotting with troubleshooting strategies

20世纪70年代末看到的第一个公开报道使用免疫印迹，评估的存在和复杂生物样品中的特定蛋白质的相对丰度的技术的。此后，免疫印迹方法已成为分子生物学家的实验剧目的共同组成部分。表明，超过22万的手稿出版已在2014年取得了重要的应用这一技术的考研用的术语"免疫印迹"粗略搜索，在过去十年里，再加上技术的发展进步成像敏感的荧光标记物具有改进的灵敏度，并且得到线性检测的更大的范围。其结果是基于西方的印迹（QFWB）现在一个真正可量化的荧光，使生物学家进行对比分析，表达与更高的灵敏度和精确度比以往任何时候。很多"优化"印迹方法存在，被用于不同的实验室。这些通常难以实现，由于微妙的，但没有证件的程序修正的要求。该协议提供了一个既定的和强大的QFWB方法的全面描述，完整的故障排除策略。

作为QFWB灵敏度和检测的线性范围大于常规ECL检测，也有一些地方是至关重要，以确保精确的数据收集的控制措施，从而有助于有效的解释。首先，包含阳性对照样品， 如图1;其次，转移的优化，以保证从凝胶向膜的高和低分子量蛋白质的等效运动为展示在图2中 ，第三，抗体的优化，特别是二次抗体的优化往往被忽视，但它可以产生非特异性条带能够与感兴趣的蛋白（S）的正确解释干扰。参见图3。第四，它也可能是这种情况，当一个蛋白似乎检测不到的，但预计将存在，这也可以是次级抗体的问题，可以通过简单地使用一个次级rais的被校正编在不同的寄主。参见图4。第五，总蛋白标记和分析是相比于使用传统的单一蛋白（多个）被普遍表达作为内部参考标准3的一个更为健壮的，可量化的方法。许多这些单个的蛋白质被发现差异表达在神经退行性疾病的模型以及与不同的组织样本和表达的均匀性可以在相同的组织中3改变。因此，生产上样对照凝胶的将确认样品负荷的均匀性，总蛋白分析，通过比较和定量蛋白质负荷在每个泳道下，在对每一个样品测定不同分子量范围的组合时，表示标准误差为展示图6中的。重要的是，所有这些故障排除技术和控制是只一样有效的分析吨的灵敏度和一致性由操作者（ 图5）施加ools。最后这种技术适合于汽提和再探测膜具有比ECL更多灵活性因包括但不限于增加的敏感性，降低的背景，双色检测和膜稳定性长期储存条件下的因素。 见图7。

Watch this Scientific Journal Video about A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies at JoVE.com

A Guide to Modern Quantitative Fluorescent Western Blotting 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 ...

a-guide-to-modern-quantitative-fluorescent-western-blotting-with

Research

JoVE Journal

Biology

51.0K 次观看.  University of Edinburgh. 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.

视频: A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

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 ...

科研

生物学

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 ...

生物化学

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

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 abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

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 ...

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 ...

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 ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

研究蛋白质精氨酸甲基转移酶1-9细胞活性的方法

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

基于免疫沉淀的技术:使用阿加玫瑰珠纯化内源性蛋白质

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

教育

JoVE Science Education

高级生物学

免疫学

Quantitative Western Blotting to Estimate Protein Expression Levels

Source: Huang, Y. T., et al. <a href="https://app.jove.com/v/59438">Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting.</a> J. Vis. Exp. (2019).
The video demonstrates a total protein normalization-based western blotting technique for quantifying the target protein expression in a sample. By utilizing the total protein staining of the sample, a ratiometric analysis is performed to determine the relative expression level of the target protein.