Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3798-2_5
Angy Liseth Davalos Macias, Lilian Costa Alecrim, Fabio C L Almeida, Ricardo Jose Giordano
Protein-protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein-protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide-protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.
{"title":"Understanding the Structural Requirements of Peptide-Protein Interaction and Applications for Peptidomimetic Development.","authors":"Angy Liseth Davalos Macias, Lilian Costa Alecrim, Fabio C L Almeida, Ricardo Jose Giordano","doi":"10.1007/978-1-0716-3798-2_5","DOIUrl":"10.1007/978-1-0716-3798-2_5","url":null,"abstract":"<p><p>Protein-protein interaction is at the heart of most biological processes, and small peptides that bind to protein binding sites are resourceful tools to explore and understand the structural requirements for these interactions. In that sense, phage display is a well-suited technology to study protein-protein interactions, as it allows for unbiased screening of billions of peptides in search for those that interact with a protein binding domain. Here, we will illustrate how two distinct but complementary approaches, phage display and nuclear magnetic resonance (NMR), can be utilized to unveil structural details of peptide-protein interaction. Finally, knowledge derived from phage mutagenesis and NMR studies can be streamlined for quick peptidomimetic design and synthesis using the retroinversion approach to validate using in vitro and in vivo assays the therapeutic potential of peptides identified by phage display.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140288503","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3738-8_14
Haw-Young Kwon, Young-Tae Chang, Nam-Young Kang
The development of small-molecule fluorescent probes for specific immune cell identification offers an economical alternative to expensive antibodies. Moreover, it enables the identification of live target cells and provides insights into the distinct properties of cells, leveraging their specific staining mechanisms. This chapter presents a comprehensive elucidation of the methodology employed for screening fluorescent compounds using flow cytometry measurements. A novel analytical approach is proposed to distinguish a fluorescent compound with a specific carbon length for B lymphocytes, involving an assessment of the staining index and the predominant ratio of immune cells. Moreover, a protocol is presented for investigating the staining mechanisms of these probes by employing cell mimicking models such as small unilamellar vesicles (SUVs).
用于特异性免疫细胞鉴定的小分子荧光探针的开发为昂贵的抗体提供了一种经济的替代品。此外,它还能识别活的靶细胞,并利用细胞的特异染色机制深入了解细胞的不同特性。本章全面阐述了利用流式细胞仪测量筛选荧光化合物的方法。本章提出了一种新颖的分析方法,用于区分具有特定碳长的 B 淋巴细胞荧光化合物,包括评估染色指数和免疫细胞的主要比例。此外,还介绍了一种利用细胞模拟模型(如小单酰胺囊泡 (SUV))研究这些探针染色机制的方案。
{"title":"Discovery of Live Cell Selective Fluorescent Probes and Elucidation of Their Mechanisms: Case Study of B Cell Selective Probe CDgB.","authors":"Haw-Young Kwon, Young-Tae Chang, Nam-Young Kang","doi":"10.1007/978-1-0716-3738-8_14","DOIUrl":"10.1007/978-1-0716-3738-8_14","url":null,"abstract":"<p><p>The development of small-molecule fluorescent probes for specific immune cell identification offers an economical alternative to expensive antibodies. Moreover, it enables the identification of live target cells and provides insights into the distinct properties of cells, leveraging their specific staining mechanisms. This chapter presents a comprehensive elucidation of the methodology employed for screening fluorescent compounds using flow cytometry measurements. A novel analytical approach is proposed to distinguish a fluorescent compound with a specific carbon length for B lymphocytes, involving an assessment of the staining index and the predominant ratio of immune cells. Moreover, a protocol is presented for investigating the staining mechanisms of these probes by employing cell mimicking models such as small unilamellar vesicles (SUVs).</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140288526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3738-8_9
Joseph D Tario, Kah Teong Soh, Paul K Wallace, Katharine A Muirhead
High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue® Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the "know your dye" and "know your cytometer" principles described herein.
{"title":"Monitoring Cell Proliferation by Dye Dilution: Considerations for Panel Design.","authors":"Joseph D Tario, Kah Teong Soh, Paul K Wallace, Katharine A Muirhead","doi":"10.1007/978-1-0716-3738-8_9","DOIUrl":"10.1007/978-1-0716-3738-8_9","url":null,"abstract":"<p><p>High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue<sup>®</sup> Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the \"know your dye\" and \"know your cytometer\" principles described herein.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140288533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3794-4_1
Denis O Omelchenko, Elena S Glagoleva, Anna Y Stepanova, Maria D Logacheva
This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2 mg/L and kinetin 2 mg/L; for shoot initiation 6-benzylaminopurine 2 mg/L, kinetin 0.2 mg/L, and indole-3-acetic acid 0.2 mg/L; for shoot multiplication 6-benzylaminopurine 3 mg/L and indole-3-acetic acid 0.2 mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1 mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.
{"title":"Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.","authors":"Denis O Omelchenko, Elena S Glagoleva, Anna Y Stepanova, Maria D Logacheva","doi":"10.1007/978-1-0716-3794-4_1","DOIUrl":"10.1007/978-1-0716-3794-4_1","url":null,"abstract":"<p><p>This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2 mg/L and kinetin 2 mg/L; for shoot initiation 6-benzylaminopurine 2 mg/L, kinetin 0.2 mg/L, and indole-3-acetic acid 0.2 mg/L; for shoot multiplication 6-benzylaminopurine 3 mg/L and indole-3-acetic acid 0.2 mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1 mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140293949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3794-4_13
Marta Hornyák, Agnieszka Płażek
The chlorophyll a fluorescence measurement method is used to determine the efficiency of the photosynthetic apparatus and to assess the physiological state of photosynthetic organisms. The measurement is simple, fast, and noninvasive. It is a precise tool to study photosynthesis response under stress conditions or to assess the impact of specific environmental factors on plants. Here we describe the usage of this method in environmental-controlled plant production systems differing in temperature or light source on the growth and development of common buckwheat.
叶绿素 a 荧光测量法用于确定光合装置的效率和评估光合生物的生理状态。这种测量方法简单、快速、无损伤。它是研究压力条件下光合作用反应或评估特定环境因素对植物影响的精确工具。在此,我们介绍了在温度或光源不同的环境控制植物生产系统中使用这种方法对普通荞麦生长发育的影响。
{"title":"Measurement of the Kinetics of Chlorophyll a Fluorescence by an LED-Light Source Fluorimeter, Handy PEA.","authors":"Marta Hornyák, Agnieszka Płażek","doi":"10.1007/978-1-0716-3794-4_13","DOIUrl":"10.1007/978-1-0716-3794-4_13","url":null,"abstract":"<p><p>The chlorophyll a fluorescence measurement method is used to determine the efficiency of the photosynthetic apparatus and to assess the physiological state of photosynthetic organisms. The measurement is simple, fast, and noninvasive. It is a precise tool to study photosynthesis response under stress conditions or to assess the impact of specific environmental factors on plants. Here we describe the usage of this method in environmental-controlled plant production systems differing in temperature or light source on the growth and development of common buckwheat.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140293955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3794-4_15
Monika Kula-Maximenko, Marta Hornyák, Agnieszka Płażek
Light is one of the main signals detected by plants that influence plant growth, development, and function. The light features that influence plants are the photoperiod, light intensity, and spectral composition. Manipulating light intensity and spectrum to obtain better plant growth and quality has become a popular research object in recent years. Here we describe the usage of the spectrometer Lighting Passport Pro to determine the impact of light intensity and share of individual waves in its spectrum in environment-controlled plant production systems on the growth, development, and soluble carbohydrate and phenolic synthesis of common buckwheat.
光是植物检测到的影响植物生长、发育和功能的主要信号之一。影响植物的光照特征包括光周期、光照强度和光谱成分。操纵光强和光谱以获得更好的植物生长和质量已成为近年来的热门研究对象。在此,我们介绍在环境控制的植物生产系统中使用光谱仪 Lighting Passport Pro 测定光照强度及其光谱中各个波段的比例对普通荞麦的生长、发育、可溶性碳水化合物和酚类合成的影响。
{"title":"Measurement of the Light Intensity and Spectrum Influence on Plant Growth and Secondary Metabolites of Common Buckwheat.","authors":"Monika Kula-Maximenko, Marta Hornyák, Agnieszka Płażek","doi":"10.1007/978-1-0716-3794-4_15","DOIUrl":"10.1007/978-1-0716-3794-4_15","url":null,"abstract":"<p><p>Light is one of the main signals detected by plants that influence plant growth, development, and function. The light features that influence plants are the photoperiod, light intensity, and spectral composition. Manipulating light intensity and spectrum to obtain better plant growth and quality has become a popular research object in recent years. Here we describe the usage of the spectrometer Lighting Passport Pro to determine the impact of light intensity and share of individual waves in its spectrum in environment-controlled plant production systems on the growth, development, and soluble carbohydrate and phenolic synthesis of common buckwheat.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140293956","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3794-4_8
Reneé Pérez-Pérez, Jolanta Kwasniewska
This chapter presents the squash chromosome preparation technique for Fagopyrum esculentum and F. tataricum, using the root tips as the source of the material. Using an optimized version of this method, the chromosomes are free of cytoplasmic debris and are spread evenly on the glass slide. What comes of it is the possibility to make observations of the chromosome number and structure at the metaphase stage. This technique's modified version allows micronuclei analysis in interphase cells of buckwheats.
本章介绍了以根尖为材料来源制备 Fagopyrum esculentum 和 F. tataricum 的壁球染色体的技术。使用该方法的优化版本,染色体不含细胞质碎片,并能均匀地铺在玻璃载玻片上。这样做的结果是,可以在分裂期观察染色体的数量和结构。这种技术的改进版可以对荞麦的间期细胞进行微核分析。
{"title":"Visualization of Fagopyrum esculentum and Fagopyrum tataricum Chromosomes and Micronuclei.","authors":"Reneé Pérez-Pérez, Jolanta Kwasniewska","doi":"10.1007/978-1-0716-3794-4_8","DOIUrl":"10.1007/978-1-0716-3794-4_8","url":null,"abstract":"<p><p>This chapter presents the squash chromosome preparation technique for Fagopyrum esculentum and F. tataricum, using the root tips as the source of the material. Using an optimized version of this method, the chromosomes are free of cytoplasmic debris and are spread evenly on the glass slide. What comes of it is the possibility to make observations of the chromosome number and structure at the metaphase stage. This technique's modified version allows micronuclei analysis in interphase cells of buckwheats.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140293963","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3621-3_8
Teng Teng, Min Yu
Cancer is a common health problem with more than 90% of deaths due to metastases. Circulating tumor cells (CTCs) contain precursors that can initiate metastases. However, CTCs are rare, heterogeneous, and difficult to expand in culture. We have previously created CTC-derived cell lines from stage IV breast cancer patients. These CTC lines were used to establish single-cell CTC clones using flow cytometry cell sorting.
{"title":"Establishing Single-Cell Clones from In Vitro-Cultured Circulating Tumor Cells.","authors":"Teng Teng, Min Yu","doi":"10.1007/978-1-0716-3621-3_8","DOIUrl":"https://doi.org/10.1007/978-1-0716-3621-3_8","url":null,"abstract":"<p><p>Cancer is a common health problem with more than 90% of deaths due to metastases. Circulating tumor cells (CTCs) contain precursors that can initiate metastases. However, CTCs are rare, heterogeneous, and difficult to expand in culture. We have previously created CTC-derived cell lines from stage IV breast cancer patients. These CTC lines were used to establish single-cell CTC clones using flow cytometry cell sorting.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139403609","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3702-9_5
Ying Zhang, Xinyue Fan, Xiaofei Cheng
Double-stranded RNA (dsRNA) is the replicate intermediate of all RNA viruses, and is also recognized by their host cells as a virus-invading molecule signal. Analysis of the localization and dynamic of virus-induced dsRNA can reveal crucial information concerning the molecular mechanism of virus replication and host responses to viral infection. In this chapter, we provide an easy and efficient protocol called dsRNA binding-dependent fluorescence complementation (dRBFC) assay for labeling the dsRNAs in living plant cells using two different plant RNA viruses, namely potato virus X and turnip mosaic virus. Moreover, both YFP- and mRFP-based dRBFC plasmids are available for the flexibility of experiment design.
{"title":"Analysis of Virus-Induced Double-Stranded RNA in Living Plant Cells by the dRBFC Assay.","authors":"Ying Zhang, Xinyue Fan, Xiaofei Cheng","doi":"10.1007/978-1-0716-3702-9_5","DOIUrl":"10.1007/978-1-0716-3702-9_5","url":null,"abstract":"<p><p>Double-stranded RNA (dsRNA) is the replicate intermediate of all RNA viruses, and is also recognized by their host cells as a virus-invading molecule signal. Analysis of the localization and dynamic of virus-induced dsRNA can reveal crucial information concerning the molecular mechanism of virus replication and host responses to viral infection. In this chapter, we provide an easy and efficient protocol called dsRNA binding-dependent fluorescence complementation (dRBFC) assay for labeling the dsRNAs in living plant cells using two different plant RNA viruses, namely potato virus X and turnip mosaic virus. Moreover, both YFP- and mRFP-based dRBFC plasmids are available for the flexibility of experiment design.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139570711","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-01-01DOI: 10.1007/978-1-0716-3670-1_11
Giorgia Radicioni, Mehmet Kesimer
Mucins MUC5AC and MUC5B are large glycoproteins that play an essential role in the innate defense of epithelial surfaces and their quantitation in biological samples would be informative about the health status of the tissue/samples they are derived from. However, they are difficult to study and quantify with traditional methods such as ELISA and western blot, due to their size, heterogeneity, and high degree of glycosylation. We successfully implemented a stable isotope labeling mass spectrometry approach for absolute quantification of mucin macromolecules. Here, in detail, we describe this accurate and sensitive liquid chromatography and mass spectrometry (LC-MS) method applied for both MUC5AC and MUC5B quantification in diverse and complex biological samples.
{"title":"Quantitation of MUC5AC and MUC5B by Stable Isotope Labeling Mass Spectrometry.","authors":"Giorgia Radicioni, Mehmet Kesimer","doi":"10.1007/978-1-0716-3670-1_11","DOIUrl":"10.1007/978-1-0716-3670-1_11","url":null,"abstract":"<p><p>Mucins MUC5AC and MUC5B are large glycoproteins that play an essential role in the innate defense of epithelial surfaces and their quantitation in biological samples would be informative about the health status of the tissue/samples they are derived from. However, they are difficult to study and quantify with traditional methods such as ELISA and western blot, due to their size, heterogeneity, and high degree of glycosylation. We successfully implemented a stable isotope labeling mass spectrometry approach for absolute quantification of mucin macromolecules. Here, in detail, we describe this accurate and sensitive liquid chromatography and mass spectrometry (LC-MS) method applied for both MUC5AC and MUC5B quantification in diverse and complex biological samples.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139723306","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}