Bio-protocol最新文献

{"title":"Measuring Anti-aging Effects in <i>Drosophila</i>.","authors":"Hyun-Jin Na, Joung-Sun Park","doi":"10.21769/BioProtoc.5305","DOIUrl":"10.21769/BioProtoc.5305","url":null,"abstract":"<p><p>One of the major factors contributing to aging and age-related diseases is the well-understood decline in the function of adult stem cells. Quantifying the degree of aging in adult stem cells is essential for advancing anti-aging mechanisms and developing anti-aging agents. However, no systematic approach to this exists. In this study, we developed a method to quantitatively assess the degree of aging in adult intestinal stem cells using a <i>Drosophila</i> midgut model and two aging markers. First, aging was induced in <i>Drosophila</i> with the desired genotype, and the anti-aging agent was administered 7 days before dissection. Then, the levels of two intestinal stem cell aging markers found in <i>Drosophila</i> (PH3 and γ-tubulin) were measured using immunohistochemistry. Finally, fluorescence microscopy was employed to count the number of aging markers and take images, which were analyzed using image analysis software. Using this approach, we quantitatively analyzed the effects of anti-aging agents on the aging of adult intestinal stem cells. This methodology is expected to significantly expedite the development of anti-aging agents and substantially reduce the research costs associated with aging-related studies. Key features • PH3 and γ-tubulin serve as reliable markers for quantitatively assessing aging in <i>Drosophila</i> intestinal stem cells. • This method for discovering anti-aging agents involves processes such as aging induction, treatment with anti-aging agents, dissection, fixation, antibody staining, and analysis of the results. • Vitamin D, similar to metformin and β-hydroxybutyrate, is an anti-aging agent. • Quantitative analysis of adult stem cell aging will enable the rapid and accurate identification of anti-aging agents and efficacy validation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5305"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067307/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144060325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proteome Birthdating: A Single-Sample Approach for Measuring Global Turnover Dynamics and \"Protein Age\".","authors":"Michael E Meadow, Sarah Broas, Margaret Hoare, Maria Ahmed, Fatemeh Alimohammadi, Kevin A Welle, Kyle Swovick, Jennifer R Hryhorenko, Anushka Jain, John C Martinez, Andrei Seluanov, Vera Gorbunova, Abigail Buchwalter, Sina Ghaemmaghami","doi":"10.21769/BioProtoc.5296","DOIUrl":"10.21769/BioProtoc.5296","url":null,"abstract":"<p><p>Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To facilitate such studies, we recently developed a technique termed \"proteome birthdating\" that differentially labels proteins based on their time of synthesis. Proteome birthdating enables analyses of age distributions of the proteome by tandem mass spectrometry (LC-MS/MS) and provides a methodology for investigating the protein age selectivity of diverse cellular pathways. Proteome birthdating can also provide measurements of protein turnover kinetics from single, sequentially labeled samples. Here, we provide a practical guide for conducting proteome birthdating in in vitro model systems. The outlined workflow covers cell culture, isotopic labeling, protein extraction, enzymatic digestion, peptide cleanup, mass spectrometry, data processing, and theoretical considerations for interpretation of the resulting data. Key features • Proteome birthdating barcodes the proteome with isotopically labeled precursors based on time of synthesis or \"age.\" • Global protein turnover kinetics can be analyzed from single, sequentially labeled biological samples. • Protein age distributions of subsets of the proteome can be analyzed (e.g., ubiquitinated proteins). • Age selectivity of protein properties, cellular pathways, or disease states can be investigated.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5296"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067312/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144055200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"From Colonization to High Production and <i>Plasmodium vivax</i> Infection of <i>Anopheles darlingi</i> and <i>Anopheles deaneorum</i>: a Platform for Malaria Research.","authors":"Maisa S Araujo, Alice O Andrade, Alessandra S Bastos, Najara Akira C Santos, José Daniel C Pontual, Jéssica E Araújo, Marina L Rocha, Maria Eduarda R Miguel, Ana Eliza M Costa, Joseph M Vinetz, Ricardo T Gazzinelli, Jansen F Medeiros","doi":"10.21769/BioProtoc.5302","DOIUrl":"10.21769/BioProtoc.5302","url":null,"abstract":"<p><p>The mass rearing of anopheline mosquitoes under laboratory conditions is essential for advancing malaria research. It facilitates in-depth studies on mosquito biology, behavior, and genetics and their role in <i>Plasmodium</i> transmission. However, the colonization of Neotropical anophelines such as <i>Anopheles darlingi</i>-a primary malaria vector in the Amazon region-has proven particularly challenging due to its unique reproductive characteristics. Unlike other species that can initially be colonized using forced copulation methods and later adapt to natural mating, <i>An. darlingi</i> does not copulate under forced conditions. Recent breakthroughs in <i>An. darlingi</i> colonization have been achieved using flashlight induction techniques, which have enabled the establishment and maintenance of stable laboratory populations. These advancements have created new opportunities for vector control studies in Brazil, including the testing of innovative control methods and <i>Plasmodium</i> transmission-blocking strategies. This protocol offers a comprehensive, step-by-step guide for initiating and scaling up large laboratory colonies of <i>An. darlingi</i> and <i>An. deaneorum</i>, a secondary malaria vector. It details methods for copulation induction, colony management, and successful artificial infection of mosquitoes with <i>Plasmodium vivax</i>. The guide serves as a critical resource for establishing new Neotropical anopheline colonies from different populations, contributing to future malaria research and control efforts in the Amazon. Additionally, the establishment of Brazil's first Malaria Vector Production and Infection Platform (<i>Plataforma de Produção e Infecção de Vetores da Malária</i>, PIVEM) has further supported the development of new control technologies and the study of <i>P. vivax-Anopheles</i> interaction, advancing efforts to combat malaria in the region. Key features • High production and experimental infection of <i>Anopheles</i> by <i>Plasmodium vivax.</i> <b>This protocol is used in:</b> Rev Soc Bras Med Trop (2019), DOI: 10.1590/0037-8682-0159-2019; Mem Inst Oswaldo Cruz (2020), DOI: 10.1590/0074-02760200070; Front Microbiol (2022), DOI: 10.3389/fmicb.2022.971083; Sci Rep (2023), DOI: 10.1038/s41598-023-44556-y; Am J Trop Med Hyg (2024), DOI: 10.4269/ajtmh.23-0349.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5302"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067303/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144014382","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TurboID Labeling and Analysis of Proteins in the Primary Cilium.","authors":"Xiaoliang Liu, Xuecai Ge","doi":"10.21769/BioProtoc.5303","DOIUrl":"10.21769/BioProtoc.5303","url":null,"abstract":"<p><p>Known as the cell's antenna and signaling hub, the primary cilium is a hair-like organelle with a few micrometers in length and 200-300 nm in diameter. Due to the small size of the primary cilium, it is technically challenging to profile ciliary proteins from mammalian cells. Traditional methods, such as physical isolation of cilia, are susceptible to contamination from other cellular components. Other proximity-based labeling methods via APEX or BioID have been used to map ciliary proteins. However, these approaches have their inherent limitations, including the use of toxic reagents like H₂O₂ and prolonged labeling kinetics. Here, we show a new proximity-based labeling technique for primary cilia with TurboID. TurboID presents a distinct advantage over BioID and APEX2 due to its expedited labeling kinetics, taking minutes instead of hours, and its use of a non-toxic biotin substrate, which eliminates the need for H₂O₂. When targeted to the cilium, TurboID selectively labels ciliary proteins with biotin. The biotinylated proteins are then enriched with streptavidin beads and labeled with tandem mass tags (TMT), followed by mass spectrometry (MS) detection. This protocol eliminates the requirement of toxic labeling reagents and significantly reduces the labeling time, thus providing advantages in mapping signaling proteins with high temporal resolution in live cells. Key features • Compared to other proximity labeling enzymes, TurboID offers fast labeling kinetics and uses cell-permeable biotin as the labeling reagent [1]. • This protocol includes a straightforward subcellular fractionation step to remove the nuclei to reduce the non-specific background. • This protocol has been successfully applied to the NIH 3T3 cell line and could also be applied in other cell lines and animal tissues.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5303"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067308/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Image-Based Lignin Detection in Nematode-Induced Feeding Sites in <i>Arabidopsis</i> Roots.","authors":"Muhammad Amjad Ali, Krzysztof Wieczorek","doi":"10.21769/BioProtoc.5301","DOIUrl":"10.21769/BioProtoc.5301","url":null,"abstract":"<p><p>Cyst and root-knot nematodes are sedentary biotrophic parasites that infect a wide range of plant species, causing significant annual yield and economic losses. Cyst nematodes (genera <i>Heterodera</i> and <i>Globodera</i>) induce specialized feeding structures called syncytia in host plant roots, while root-knot nematodes (<i>Meloidogyne</i> spp.) form galls containing feeding cells known as giant cells. This protocol describes the visualization of lignin in <i>Arabidopsis</i> roots infected by beet cyst nematode <i>H. schachtii</i> and root-knot nematode <i>M. incognita</i> using histochemical staining. We present two distinct approaches for lignin detection: direct staining of root segments containing syncytia and galls and histopathological detection in thin longitudinal sections of the feeding sites. Key features • First approach: Staining of intact roots visualizes lignin in nematode feeding sites and requires only simple specimen preparation and staining solution, with no sectioning needed. • Second approach: Staining of longitudinal sections of feeding sites visualizes cell-specific lignin localization and requires moderate tissue preparation and sectioning. • Both approaches enable specific detection and visualization of lignin in nematode-infected <i>Arabidopsis</i> tissues. Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5301"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067305/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144060445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reconstruction of Single-Neuron Projectomes in Mice.","authors":"Biyu Ren, Xiaoxue Shi, Bingqing Zhao, Chun Xu, Xiaofei Wang","doi":"10.21769/BioProtoc.5300","DOIUrl":"10.21769/BioProtoc.5300","url":null,"abstract":"<p><p>Reconstructing single-neuron projectomes is essential for mapping the mesoscopic connectome and eventually for understanding brain-wide connectivity and diverse brain functions. The combination of sparse labeling techniques and large-scale and high-resolution optical imaging technologies has been revolutionizing the brain-wide reconstruction of single-neuron morphologies, as exemplified by the dataset for over 10,100 single-neuron projectomes of hippocampal neurons. Here, we illustrate a comprehensive protocol for large-scale single-neuron reconstruction in the mouse brain. This includes key steps and examples in imaging data preprocessing, neurite tracing, and registration into a template brain. These procedures enable efficient and accurate large-scale morphological reconstruction of single neurons in the mouse brain. Key features • Rigorous and effective single-neuron reconstruction from raw imaging data. • Multi-person tracing and quality control for the accuracy of single-neuron tracing results. • Precise image registration based on landmark drawings of selected brain regions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5300"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067299/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144060820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mapping the Simultaneously Accessible and ssDNA-Containing Genome With KAS-ATAC Sequencing.","authors":"Georgi K Marinov, William J Greenleaf","doi":"10.21769/BioProtoc.5306","DOIUrl":"10.21769/BioProtoc.5306","url":null,"abstract":"<p><p>The KAS-ATAC assay provides a method to capture genomic DNA fragments that are simultaneously physically accessible and contain single-stranded DNA (ssDNA) bubbles. These are characteristic features of two of the key processes involved in regulating and expressing genes-on one hand, the activity of <i>cis</i>-regulatory elements (cREs), which are typically devoid of nucleosomes when active and occupied by transcription factors, and on the other, the association of RNA polymerases with DNA, which results in the presence of ssDNA structures. Here, we present a detailed protocol for carrying out KAS-ATAC as well as basic processing of KAS-ATAC datasets and discuss the key considerations for its successful application. Key features • Allows mapping of simultaneously accessible and ssDNA-containing DNA fragments. • Describes the execution of N3-kethoxal labeling and transposition of native chromatin. • Describes the pulldown of biotin-labeled DNA fragments and library generation. • Describes basic KAS-ATAC data processing steps.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5306"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067302/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144055587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"GWAS Procedures for Gene Mapping in Diverse Populations With Complex Structures.","authors":"Zhen Zuo, Mingliang Li, Defu Liu, Qi Li, Bin Huang, Guanshi Ye, Jiabo Wang, You Tang, Zhiwu Zhang","doi":"10.21769/BioProtoc.5284","DOIUrl":"https://doi.org/10.21769/BioProtoc.5284","url":null,"abstract":"<p><p>With reduced genotyping costs, genome-wide association studies (GWAS) face more challenges in diverse populations with complex structures to map genes of interest. The complex structure demands sophisticated statistical models, and increased marker density and population size require efficient computing tools. Many statistical models and computing tools have been developed with varied properties in statistical power, computing efficiency, and user-friendly accessibility. Some statistical models were developed with dedicated computing tools, such as efficient mixed model analysis (EMMA), multiple loci mixed model (MLMM), fixed and random model circulating probability unification (FarmCPU), and Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK). However, there are computing tools (e.g., GAPIT) that implement multiple statistical models, retain a constant user interface, and maintain enhancement on input data and result interpretation. In this study, we developed a protocol utilizing a minimal set of software tools (BEAGLE, BLINK, and GAPIT) to perform a variety of analyses including file format conversion, missing genotype imputation, GWAS, and interpretation of input data and outcome results. We demonstrated the protocol by reanalyzing data from the Rice 3000 Genomes Project and highlighting advancements in GWAS model development.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5284"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021685/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026565","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Antibody Purification Using Spy Chemistry.","authors":"Xiaofeng Yang, Zhanglin Lin, Ya Xiang, Zisha Lao","doi":"10.21769/BioProtoc.5286","DOIUrl":"https://doi.org/10.21769/BioProtoc.5286","url":null,"abstract":"<p><p>Antibody purification is a fundamental technology for therapeutic and diagnostic applications. While traditional methods like ammonium sulfate precipitation and polyethylene glycol precipitation are cost-effective, they often result in low purity and require multiple purification steps. Protein A-based affinity chromatography, the gold standard for antibody purification, provides high specificity but can be further improved to increase loading capacity and reduce costs. In this protocol, we introduce a novel approach for purifying high-quality, high-purity antibodies from complex samples using SpyFixer/Z domain-modified resin. This method utilizes Spy chemistry for site-specific immobilization of the Z domain of Protein A, significantly enhancing antibody loading capacity up to 200 mg/mL resin and ensuring stable, durable immobilization. Using this protocol, we achieved >90% purity of human immunoglobulin G (hIgG) from diverse sources, including <i>E. coli</i> cell lysates, human serum, and industrial fermentation broth. The SpyFixer/Z domain-modified resin protocol is simple, cost-effective, and offers a robust, scalable solution for efficient antibody purification in bioengineering applications. This immobilization scheme based on Spy chemistry can also be extended to other immunoglobulin-binding proteins, such as Protein G and Protein L, to develop high-efficiency affinity resins. Key features • This protocol builds upon purification methods developed by Lin's lab [1], providing more detailed steps than the previously published study. • The protocol offers a useful and standardized approach for purifying antibodies and Fc-fusion proteins. • The SpyFixer/Z domain-modified resin is easy to prepare, reusable, and offers a cost-effective solution.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5286"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021679/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144033965","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Visualization of F-Actin Through Expansion Microscopy (ExM) with Trifunctional Linker-Conjugated Phalloidin.","authors":"Jianjun Huang, Gang Wen, Thibo Iven, Débora Linhares, Leewon Koo, Markus Sauer, Wim Dehaen, Volker Leen, Johan Hofkens","doi":"10.21769/BioProtoc.5273","DOIUrl":"https://doi.org/10.21769/BioProtoc.5273","url":null,"abstract":"<p><p>Expansion microscopy (ExM) is an imaging technique that enables super-resolution imaging of biological specimens using conventional confocal microscopy. This process entails the isotropic physical expansion of a (biomolecular) sample that has been cross-linked to a swellable polymer. The grafting of biomolecules (and the subsequent fluorescent readout) is accomplished by introducing an acryloyl group to the amine groups of lysine residues within the proteins, enabling subsequent imaging. However, visualizing actin filaments with high spatial resolution using ExM remains challenging. Herein, we report the construction of a phalloidin conjugate containing actin stains and their application in ExM. This protocol highlights the efficacy of trifunctional linker (TRITON/Actin-ExM) for F-actin imaging, demonstrating that TRITON-labeled actin allows for efficient anchoring and signal retention, enabling robust visualization of actin filaments in expansion microscopy. Key features • Engineered linker (TRITON) design ensures efficient fluorophore attachment, resulting in bright, stable signals during imaging. • Performed pre-expansion and antibody-free labeling. • Detailed and specific visualization of actin filaments in ExM experiments (4-fold expansion).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 8","pages":"e5273"},"PeriodicalIF":1.0,"publicationDate":"2025-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12021582/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144002325","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}