1.抗体捕获到高容量磁蛋白A或磁G蛋白珠<ol><li>均匀再通过暂停轻轻摇动磁珠。保持均匀的悬浮液珠等分的时候。 </li><li>加入50μl珠浆料到1.5ml微量离心管中。在磁力架，持续10秒的地方。小心取出存储缓冲区。 </li><li>加入250微升抗体结合缓冲。 </li><li>混合并在磁性支架10秒的地方。小心取出结合缓冲液。 </li><li> ，增加1.0毫升含50-100微克抗体与珠粒样品。样品可被纯化在细胞培养基的抗体或抗体。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。 </li><li>放置管在磁性支架10秒并除去上清液。 </li><li>加入250微升抗体结合/洗涤液混合。在磁性支架放置10秒，取出结合/洗涤缓冲液。重复这个STEP总共两次洗涤的。 </li><li>继续第2节使用胺化学或第3节使用硫醇化学标签抗体标签抗体。 </li></ol> 2.抗体标记使用胺化学<ol><li>共轭抗体<ol><li>添加100μl的胺偶联缓冲液中的珠。 </li><li>通过加入100微升二甲基亚砜（DMSO）中的至1.0mg染料溶解在10毫克/毫升的胺反应性的荧光染料（AlexaFluor 532-SE）。震荡混合。使该解决方案使用前。 </li><li>添加2.5微升的胺 - 反应性染料为100微克的抗体。 注意:通常建议一个5-20摩尔过量活性染料。然而，反应性染料的量加入到反应需要根据所需染料与抗体比例和特性的抗体性质来凭经验优化。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。确保珠留在悬索桥ñ。 </li><li>放置管在磁性支架10秒并除去上清液。 </li><li>加入250微升抗体结合/洗涤液混合。在磁力架，持续10秒的地方。取下并丢弃绑定/洗涤液。重复此步骤，总共两次洗涤。 </li></ol></li><li>抗体恢复<ol><li>加入50μl洗脱缓冲液至珠。 </li><li>使用涡旋混合器或最终过端混合器在RT混合5分钟。确保珠粒留在悬浮液中。 </li><li>放置管在磁性支架10秒。除去洗脱样品，并转移到含有5微升中和缓冲液的一个新的微离心管中。 </li><li>重复这个过程更多的时间，集中洗脱的样品。 </li><li>定量抗体浓度，并如第4所述的染料与抗体的比例。 </li></ol></li></ol> 3.抗体标记使用硫醇化学<ol><li>减少抗体<ol><li>加入250微升巯基结合缓冲和组合。放置管在磁性支架10秒。取下并丢弃缓冲区。重复此步骤两次。 </li><li>加入100微升巯基结合缓冲。 </li><li>添加二硫苏糖醇（DTT）至2.5mM的终浓度。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。确保珠粒留在悬浮液中。 </li><li>放置管在磁性支架10秒，并丢弃缓冲器。 </li><li>加入250微升巯基结合缓冲液混匀。放置管在磁性支架10秒。取下并丢弃缓冲区。重复此步骤，总共两次洗涤。 </li><li>加入100微升巯基结合缓冲。 </li></ol></li><li>共轭抗体<ol><li>通过加入100微升的DMSO至1.0mg染料溶解在10毫克/毫升的硫醇反应性染料。震荡混合。使该解决方案使用前。 </li><li>添加2.5微升硫醇反应性染料为100微克抗身体。 注意:通常建议一个5-20摩尔过量活性染料。然而，反应性染料的量加入到反应需要根据所需染料与抗体比例和特性的抗体性质来凭经验优化。 </li><li>使用涡旋混合器或最终过端混合器在RT混合样品60分钟。确保珠粒留在悬浮液中。 </li><li>把试管磁力架，持续10秒，取出上清。 </li><li>加入250微升巯基结合缓冲液混匀。在磁保持10秒的地方，去掉缓冲区。重复此步骤，总共两次洗涤。 </li></ol></li><li>洗脱抗体<ol><li>加入50μl洗脱缓冲液至珠。 </li><li>使用涡旋混合器或最终过端混合器在RT混合5分钟。确保珠粒留在悬浮液中。 </li><li>放置管在磁性支架10秒。删除洗脱样品，并转移到含有5微升NEUT的一个新的微离心管ralization缓冲区。 </li><li>重复这个过程更多的时间，集中洗脱的样品。 </li><li>定量抗体浓度，并如第4所述的染料与抗体的比例。 </li></ol></li></ol> 4.计算染料对抗体的比率<ol><li>测量抗体 - 染料偶联物的280nm处的吸光度（A280），并在λ最大为染料（安迈）。 </li><li>计算抗体浓度: 抗体浓度（毫克/毫升）= A280 - （A× 最大 CF）/ 1.4 其中，CF染料（由生产商提供的）的=校正因子。 </li><li>计算染料与抗体的比例: 染料与抗体的比率（DAR）=（A 最大值 ×15万条）/抗体浓度（毫克/毫升）×ε 染料 式中，ε 染料 =在其最大吸光度的染料的消光系数和抗体的分子量= 150000道尔顿。 </li></ol>

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Authors are employees of Promega Corporation.

本研究的目的是开发标记抗体中，存在于在低浓度的细胞培养基，具有多种小分子的方法。这种方法将允许大量的抗体，在抗体发现的早期阶段，与小分子进行标记，并使用生物相关测定法进行筛选。一种这样的测定法是受体介导的抗体的内化测定法，其中内化可能甚至具有相似的结合亲和力的抗体之间变化。因此，为了具体地筛选抗体除了传统的酶联基于免疫吸附测定（ELISA）筛选其内在特性?...

标记的小分子抗体是也许是生物学1,2-最常用的试剂。用荧光染料标记和生物素的抗体在成像，免疫测定被广泛使用，流式细胞术，Western印迹，免疫沉淀和其他应用程序3-6中。放射性标记的抗体3,7-发现在成像和治疗，标有细胞毒性药物（ADC）的抗体广泛使用用于癌症的治疗中，提供新的选择，和两个ADC已经被批准用于治疗用途8。尽管他们广泛使用，用于标记抗体的方法仍然令人惊讶的改变，通常涉及多个反应和脱盐步骤9-12。溶液的方法中，其中只有少数的抗体需要被标记，并在高度纯化的形式提供在高浓度情况下，并且在足以卷工作得很好。然而，对于象ADC的新的应用程序，还有一个需要在早期杂交瘤细胞阶段来标记抗体，使它们可以筛选生物相关的属性，例如，受体介导的抗体的内化13-16。在杂交瘤阶段，样品体积是有限的，抗体在低浓度下表达和若干样本都很大，因此在基于溶液的标记方法是不适合的。 简化和改进了传统的抗体标记方法的吞吐量，一些替代办法，已经提出17,18。一种方法是使用非磁性蛋白装在小的列A亲和珠捕获抗体随后标记反应和标记的和纯化的蛋白的洗脱。这种方法可用于直接从细胞培养基标记的抗体，然而，使用列可以是费力。磁珠基于方法最近有报道19，消除了使用列和提高吞吐量但由于在有限的抗体结合的珠容量，仅纳克至抗体的低微克量可以进行标记。 我们最近开发并用于高容量磁性蛋白A和蛋白G珠（&gt; 20毫克人IgG /毫升沉降的珠子）标记存在于细胞培养基与小分子20的抗体。珠的高容量允许几十到几百抗体微克能够方便标记和珠的快速磁响应简化的大量平行样品的处理和加工。使用荧光染料作为代理人为小分子，我们表明，该方法是与胺和硫醇化学标记兼容，并提供了标记，并非常纯净抗体的高回收率。 这个协议和所附视频描述存在于使用磁性蛋白A和蛋白G珠的细胞培养基小鼠抗体的小球上的标记。该协议是分为四个部分:第一部分介绍到生物样品珠抗体捕获。继拍摄，采用胺化学或使用硫醇化学与荧光染料抗体的标签在第2和第3节分别描述。最后，第4描述了一种用于抗体浓度的计算和染料与抗体的比例的方法。

<table border="0" cellpadding="0" cellspacing="0" width="592">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein A Beads</td>
			<td style="width:103px;">Promega Corporation</td>
			<td style="width:119px;">&#160;G8781</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein G Beads</td>
			<td style="width:103px;">Promega Corporation&#160;</td>
			<td style="width:119px;">&#160;G7471</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-SE (Succinimidyl Ester)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-ME (Malemide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A10255</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 647-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20347</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescein-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">F-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic Stand</td>
			<td style="width:103px;">Promega</td>
			<td style="width:119px;">Z5332</td>
			<td></td>
		</tr>
	</tbody>
</table>

antibody labeling with fluorescent dyes using magnetic protein a and protein g beads

标有类似的荧光染料，细胞毒性药物和放射性示踪剂的小分子抗体是生物医学研究的重要工具，免疫诊断试剂以及最近作为治疗剂。对于小分子标记抗体的传统方法需要在相对高浓度的纯化抗体，涉及多个透析步骤和有限的吞吐量。然而，一些应用，包括抗体药物偶联物（ADC）的领域中，将可使用的新方法，这将允许抗体的标记直接从细胞培养基。这样的方法可以允许抗体在生物学相关测定法进行筛选，例如，在ADC的情况下，受体介导的抗体内测定。这里，我们描述的方法（小球上的方法），使少量的抗体的标记直接从细胞培养基。这种方法利用高容量磁蛋白A和G蛋白亲和珠捕获抗体从细胞培养基，随后通过标签使用任一胺或硫醇化学和标记抗体的随后洗脱的小分子。以荧光染料作为替代物的小分子，我们展示了三种不同的小鼠抗体的小球上的标签直接从同时使用胺和硫醇化学标记细胞培养基。抗体与蛋白A和蛋白G的高结合亲和力，确保高回收率，以及标记的抗体的纯度高。另外，使用的磁珠允许多个样品进行人工处理，从而显著提高标签的吞吐量。

一种使用高容量磁性蛋白A和蛋白G磁珠标记的小分子的抗体概略示于图1。抗体上的磁性蛋白G珠可与小分子标记捕获，例如荧光染料，即使用胺化学哪些标签赖氨酸氨基酸或用硫醇化学哪些标签在抗体的铰链区还原硫醇的伯胺。抗体和蛋白G之间的亲和力强21和结果在标记反应中抗体的损失最小。相比简单的纯化时，这反映在与荧光染料标记后三种不同?...

Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

上的胎圈的方法用于标记抗体与小分子使抗体的少量的标记直接从细胞培养基。这种方法是与胺和硫醇化学相容，并且可以并行处理多个样品，手工或使用自动平台。

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

antibody-labeling-with-fluorescent-dyes-using-magnetic-protein

Research

JoVE Journal

Immunology and Infection

11.8K 次观看.  Promega Corporation. 上的胎圈的方法用于标记抗体与小分子使抗体的少量的标记直接从细胞培养基。这种方法是与胺和硫醇化学相容，并且可以并行处理多个样品，手工或使用自动平台。 

视频: Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

HIV - 1使用转速依赖的慢病毒表达载体的绿色荧光蛋白阳性细胞的特定标记

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

科研

免疫与感染

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

大肠癌细胞表面蛋白的分析，使用一种抗体的芯片和荧光多路复用

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

朊病毒蛋白质错误折叠循环扩增

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

接种<em&gt;冈比亚按蚊</em&gt;珠蚊子诱发和测量黑化免疫反应

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

分子互补亲和纯化 (BiCAP) 对多蛋白信号络合物的解剖

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

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