Bio-protocol最新文献

{"title":"Rapid and Uniform NHS-Ester-Based Membrane Protein Labeling of Live Mammalian Cells.","authors":"Alyssa Burgess, Hirushi Gunasekara, Ying S Hu","doi":"10.21769/BioProtoc.5455","DOIUrl":"10.21769/BioProtoc.5455","url":null,"abstract":"<p><p>Rapid and uniform labeling of plasma membrane proteins is essential for high-resolution imaging of dynamic membrane topologies and intercellular communication in live mammalian cells. Existing strategies for labeling live cell membranes, such as fluorescent fusion proteins, enzyme-mediated tags, metabolic bioorthogonal labeling, and lipophilic dyes, face trade-offs in the requirement of genetic manipulation, the presence of non-uniform labeling, the need for extensive preparation times, and limited choices of fluorophores. Here, we present a streamlined protocol that leverages N-hydroxysuccinimide (NHS)-ester chemistry to achieve rapid (≤5 min), covalent conjugation of synthetic small-molecule dyes to surface-exposed primary amines, enabling pan-membrane-protein labeling. This workflow covers dye stock preparation, labeling for suspension and adherent cells, multiplex live-cell imaging, fusion protein co-staining (including insulin-triggered receptor endocytosis), 3D membrane visualization, and in vivo assays for visualizing membrane-derived material transfers between donor and recipient cells using a lymphoma T-cell mouse model. This high-density labeling approach is compatible with various cell types across diverse imaging platforms. Its speed, versatility, and stability make it a broadly applicable tool for studying plasma membrane dynamics and intercellular membrane trafficking. Key features • Rapid high-density membrane labeling with small-molecule fluorescent dyes. • Enables live-cell multiplexed imaging, amenable to primary cells and cells expressing fluorescent fusion proteins, and supports in vivo studies of membrane-associated cell-cell communications. • Compatible with various fluorescence imaging modalities.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5455"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514140/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282064","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 Profiling in Live Cells Using Live Cell Painting.","authors":"Thaís Moraes-Lacerda, Mariana Rodrigues-Da-Silva, Shantanu Singh, Marcelo Bispo De Jesus","doi":"10.21769/BioProtoc.5464","DOIUrl":"10.21769/BioProtoc.5464","url":null,"abstract":"<p><p>High-content analysis (HCA) is a powerful image-based approach for phenotypic profiling and drug discovery, enabling the extraction of multiparametric data from individual cells. Traditional HCA protocols often rely on fixed-cell imaging, with assays like cell painting widely adopted as standard. While these methods provide rich morphological information, the integration of live-cell imaging expands analytical capabilities by enabling the study of dynamic biological processes and real-time cellular responses. This protocol presents a simple, cost-effective, and scalable method for live-cell HCA using acridine orange (AO), a metachromatic fluorescent dye that highlights cellular organization by staining nucleic acids and acidic compartments. The assay provides visualization of distinct subcellular structures, including nuclei and cytoplasmic organelles, using a two-channel fluorescence readout. Compatible with high-throughput microscopy and computational analysis, the method supports diverse applications such as phenotypic screening, cytotoxicity assessment, and morphological profiling. By preserving cell viability and enabling dynamic, real-time measurements, this live-cell imaging approach complements existing fixed-cell assays and offers a versatile platform for uncovering complex cellular phenotypes. Key features • Builds upon Garcia-Fossa et al. [1], providing an accessible workflow for image-based profiling in live cells. • Enables phenotypic profiling and dose-response analysis of diverse perturbants, including small molecules, oligonucleotides, and nanoparticles. • Provides a live-cell framework to detect subtle, sublethal phenotypic changes, overcoming fixation assay limitations in toxicology and drug discovery. • Includes a streamlined analysis pipeline supporting efficient and reproducible interpretation of image-based data.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5464"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514141/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282130","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":"In Vitro Screening of Microbial Extracts Against the Oomycetes <i>Phytophthora capsici</i> and <i>Pythium ultimum</i>.","authors":"Mónica Trigal Martínez, Antonio Fernández Medarde, María Ángeles Vinuesa Navarro","doi":"10.21769/BioProtoc.5451","DOIUrl":"10.21769/BioProtoc.5451","url":null,"abstract":"<p><p>Oomycetes are a predominantly plant-pathogenic group of organisms often considered and managed as fungi; however, due to their evolutionary divergence from true fungi, many conventional fungicides are ineffective against them. Their unique physiological characteristics make them challenging to work with, highlighting the need for a standardized and reproducible procedure for anti-oomycete assays. Previous studies describe methods to obtain sporulation forms in the laboratory, but there remains a disconnect between spore production and the subsequent screening process for potential biological pesticides based on microbial organic extracts. This protocol bridges that gap by providing a complete and reliable workflow from spore production to screening. In this study, we present an efficient in vitro protocol to identify microbial extracts with activity against <i>Phytophthora capsici</i> and <i>Pythium ultimum.</i> The protocol includes a method for obtaining zoospores of <i>P. capsici</i> and oospores of <i>P. ultimum</i>, followed by a simple and rapid screening assay to detect microbial extracts that inhibit the growth of these pathogens. The extracts are dispensed onto plates in two concentrations and allowed to dry. This facilitates pauses in the protocol and allows for storage of the plates until the biological material is ready for the assay. The protocol's effectiveness has been validated with these two oomycetes, resulting in the identification of active extracts in both cases. Moreover, it can be adapted to other pathogens. Key features • This protocol is designed for in vitro susceptibility testing of oomycetes, using <i>Phytophthora</i> zoospores and <i>Pythium</i> oospores as inoculum sources. • This protocol can be easily used for the identification of natural compounds with anti-oomycete activity.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5451"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457833/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152354","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":"Protocol for the Preparation of a Recombinant Treacle Fragment for Liquid-Liquid Phase Separation (LLPS) Assays.","authors":"Nadezhda V Petrova, Konstantin I Balagurov, Sergey V Razin, Artem K Velichko","doi":"10.21769/BioProtoc.5439","DOIUrl":"10.21769/BioProtoc.5439","url":null,"abstract":"<p><p>Liquid-liquid phase separation (LLPS) underlies the spatial organization of the nucleolus, a membraneless organelle responsible for ribosomal RNA (rRNA) transcription and ribosome subunit assembly. One of the key proteins involved in the formation of the fibrillar center of the nucleolus is the treacle, an intrinsically disordered protein that contains low-complexity repeats enriched in charged amino acid residues. In this work, we present a detailed protocol for the bacterial expression and purification of a recombinant fragment of treacle comprising two tandem low-complexity repeat (LCR) modules, with a total length of 136 amino acids. This fragment is intended for subsequent in vitro investigation of its ability to undergo LLPS. The described method enables the production of a soluble, biochemically pure protein preparation suitable for studying the mechanisms of spontaneous condensate formation in a cell-free system. This approach allows for the controlled modeling and quantitative evaluation of the contribution of low-complexity sequences to the phase behavior of treacle, independently of its interactions with cellular partners in vivo. Key features • Protocol describes the expression and purification of a soluble treacle fragment containing two LCR motifs, suitable for in vitro LLPS studies. • Avoids complications associated with full-length treacle expression by using a minimal, well-behaved construct. • Produces biochemically pure protein compatible with phase separation assays under controlled buffer and crowding conditions. • Applicable for dissecting sequence features driving electrostatically mediated LLPS in nucleolar IDR/LCR proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5439"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457835/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152390","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":"Protocol for Quantitative Estimation of Hydrogen Cyanide Production from Bacteria.","authors":"Devashish Pathak, Pushpendra Sharma, Venkadasamy Govindasamy, Archna Suman","doi":"10.21769/BioProtoc.5441","DOIUrl":"10.21769/BioProtoc.5441","url":null,"abstract":"<p><p>Hydrogen cyanide (HCN) is a volatile, nitrogen-containing secondary metabolite produced by various bacterial species, primarily during the idiophase of growth under nutrient-limiting or competitive conditions. It plays a significant ecological role as a biocontrol agent by inhibiting the respiratory enzymes of plant pathogens and modulating microbial competition in the rhizosphere. Although protocols for detecting HCN production have existed for over a century, they have largely remained qualitative and are rarely optimized for quantitative assessment. This is mainly due to the volatile nature of HCN, unidentified stable reference standards, and the absence of a robust, universally accepted protocol that ensures consistency across diverse microbial types. In this study, we present a simplified and efficient colorimetric method to quantify HCN production in both Gram-positive and Gram-negative bacteria. Qualitatively, HCN production was observed by a color change due to the isopurpurate complex. This compound was then eluted and quantified by measuring absorbance at 625 nm. The method uses potassium ferrocyanide as a standard, whose slow dissociation constant enables a stable and controlled release of cyanide ions for calibration, unlike highly dissociative salts like KCN that introduce early volatilization errors. This protocol demonstrated high sensitivity, capable of detecting HCN at concentrations as low as ppm levels, with strong correlation to the standard curve (R² > 0.99). Achieving such sensitivity with other conventional methods, such as gas detection tubes or electrochemical sensors, often requires more sophisticated instrumentation and strict handling conditions. In contrast, this approach offers a cost-effective, reproducible, and user-friendly alternative. While a universally adopted method is still lacking due to standardization challenges and HCN volatility, the proposed protocol marks a significant advancement toward accurate and accessible quantitative assessment in microbiological and agricultural applications. Key features • Enables both qualitative detection and quantitative estimation of hydrogen cyanide (HCN) production in bacteria using a colorimetric assay. • Utilizes a low-dissociation reference compound, potassium ferrocyanide, to create a stable and accurate standard curve for reproducible measurement of HCN concentration. • Offers a simple, cost-effective, and broadly applicable method suitable for screening HCN-producing bacteria in both Gram-positive and Gram-negative groups. • Highly sensitive and accurate HCN detection at sub-ppm levels, ensuring rapid colorimetrical results and reproducibility.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5441"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152410","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":"A Protocol to Assess Time-of-Day-Dependent Learning and Memory in Mice Using the Novel Object Recognition Test.","authors":"Jordan Mar, Matthew A McGregor, Vishnuvasan Raghuraman, Isabella Farhy-Tselnicker","doi":"10.21769/BioProtoc.5446","DOIUrl":"10.21769/BioProtoc.5446","url":null,"abstract":"<p><p>Changes in learning and memory are important behavioral readouts of brain function across multiple species. In mice, a multitude of behavioral tasks exist to study learning and memory, including those influenced by extrinsic and intrinsic forces such as stress (e.g., escape from danger, hunger, or thirst) or natural curiosity and exploratory drive. The novel object recognition (NOR) test is a widely used behavioral paradigm to study memory and learning under various conditions, including age, sex, motivational state, and neural circuit dynamics. Although mice are nocturnal, many behavioral tests are performed during their inactive period (light phase, subjective night) for the convenience of the diurnal experimenters. However, learning and memory are strongly associated with the animal's sleep-wake and circadian cycles, stressing the need to test these behaviors during the animals' active period (dark phase, subjective day). Here, we develop a protocol to perform the NOR task during both light (subjective night) and dark (subjective day) phases in adult mice (4 months old) and provide a flexible framework to test the learning and memory components of this task at distinct times of day and associated activity periods. We also highlight methodological details critical for obtaining the expected behavioral responses. Key features • Enables analysis of learning and memory in mice during both active (dark) and inactive (light) phases. • Allows for switching the time-of-day-dependent familiarization and recognition to study the impact of activity and sleep-wake cycle on cognitive performance. • Details the environmental and experimenter-dependent conditions that can impact behavioral responses. • Provides a flexible, adjustable platform for testing variable experimental conditions such as age, sex, learning and memory components, and genetic manipulations.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5446"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457838/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152161","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":"CRISPR/Cas9-Induced Targeted Mutagenesis of the Moss <i>Physcomitrium patens</i> by Particle Bombardment-Mediated Transformation.","authors":"So Takenaka, Mamoru Sugita, Toshihisa Nomura, Setsuyuki Aoki","doi":"10.21769/BioProtoc.5452","DOIUrl":"10.21769/BioProtoc.5452","url":null,"abstract":"<p><p>The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a widely used programmable nuclease system for gene modification in many organisms, including <i>Physcomitrium patens. P. patens</i> is a model species of moss plants, a basal land plant group, which has been extensively studied from the viewpoint of evolution and diversity of green plant lineages. So far, gene modifications by CRISPR/Cas9 in <i>P. patens</i> have been carried out exclusively by the polyethylene glycol (PEG)-mediated DNA transfer method, in which a transgene (or transgenes) is introduced into protoplast cells prepared from protonemal tissues. However, this PEG-mediated method requires a relatively large amount of transgene DNA (typically 30 µg for a single transformation), consists of many steps, and is time-consuming. Additionally, this PEG-mediated method has only been established in a few species of moss. In the current protocol, we succeeded in CRISPR/Cas9-induced targeted mutagenesis of <i>P. patens</i> genes by making good use of the biolistic method, which i) requires amounts of transgene DNA as low as 5 μg for each vector, ii) consists of fewer steps and is time-saving, and iii) is known to be applicable to a wide variety of species of plants. Key features • In this protocol, particle bombardment-mediated gene transfer is used for CRISPR/Cas9-induced mutagenesis in the moss <i>Physcomitrium patens</i>. • By this application of particle bombardment, a gene can be modified by the CRISPR/Cas9 system much more conveniently with a smaller amount of transgene DNA. • This protocol is expected to be easily applicable to non-model moss species, some of which have noteworthy traits, such as tolerance to various stresses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5452"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457832/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152221","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":"A Protocol for Weighted Gene Co-expression Network Analysis With Module Preservation and Functional Enrichment Analysis for Tumor and Normal Transcriptomic Data.","authors":"Phuong Nguyen, Erliang Zeng","doi":"10.21769/BioProtoc.5447","DOIUrl":"10.21769/BioProtoc.5447","url":null,"abstract":"<p><p>Weighted gene co-expression network analysis (WGCNA) is widely used in transcriptomic studies to identify groups of highly correlated genes, aiding in the understanding of disease mechanisms. Although numerous protocols exist for constructing WGCNA networks from gene expression data, many focus on single datasets and do not address how to compare module stability across conditions. Here, we present a protocol for constructing and comparing WGCNA modules in paired tumor and normal datasets, enabling the identification of modules involved in both core biological processes and those specifically related to cancer pathogenesis. By incorporating module preservation analysis, this approach allows researchers to gain deeper insights into the molecular underpinnings of oral cancer, as well as other diseases. Overall, this protocol provides a framework for module preservation analysis in paired datasets, enabling researchers to identify which gene co-expression modules are conserved or disrupted between conditions, thereby advancing our understanding of disease-specific vs. universal biological processes. Key features • Presents a step-by-step WGCNA protocol with module preservation and functional enrichment analysis [1,2] using TCGA cancer data, demonstrating network differences between normal and tumor tissues. • Preprocesses gene expression data and conducts downstream analysis for constructed networks. • Requires 2-3 h hands-on time and 8-12 h total computational time, depending on dataset size and permutation number used for module preservation analysis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5447"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457846/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152208","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":"Editing the <i>Serratia proteamaculans</i> Genome Using the Allelic Exchange Method.","authors":"Ksenia Chukhontseva, Maria Karaseva, Aleksey Komissarov, Ilya Demidyuk","doi":"10.21769/BioProtoc.5448","DOIUrl":"10.21769/BioProtoc.5448","url":null,"abstract":"<p><p>No specific ecological niche has been identified for <i>Serratia proteamaculans</i>. Different strains of the bacterium have been described as opportunistic pathogens of plants, animals, and humans, as plant symbionts, and as free-living bacteria. This makes <i>S. proteamaculans</i> and its particular strains promising models for research, particularly aimed at studying the role of various genes in interspecific interactions. Genome editing is one of the most significant approaches used to study gene function. However, as each bacterial species has its own characteristics, editing methods often need to be adapted. In this study, we adapted a conventional approach based on homologous recombination-the allelic exchange method-to edit the genome of <i>S. proteamaculans</i>, with the aim of examining the biological role of protealysin. Plasmids for recombination were created using the suicidal vector pRE118, and then an auxotrophic <i>Escherichia coli</i> ST18 strain was used to deliver these plasmids to <i>S. proteamaculans</i> through conjugation. This method is valid and can potentially be used to create knockouts, knockins, and point mutations in the <i>S. proteamaculans</i> genome, without the need to insert a selective marker into the genome. Key features • The genome editing method for <i>Serratia proteamaculans</i> does not require the insertion of selective markers into the genome. • The selection strategy allows obtaining 30%-40% of clones with the target mutation at the final stage. • The method can be adapted to introduce knockouts, knockins, and point mutations into the genomes of other bacteria.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5448"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457842/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152278","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":"Fractionation and Extraction of Cell Wall Proteins From <i>Candida albicans</i>.","authors":"Elizabeth Reyna-Beltrán, María Iranzo, Salvador Mormeneo, César Isaac Bazán-Méndez, María Luisa Labra-Barrios, Edgar Hernandez-Martínez, Juan Pedro Luna-Arias","doi":"10.21769/BioProtoc.5456","DOIUrl":"10.21769/BioProtoc.5456","url":null,"abstract":"<p><p><i>Candida albicans</i> is the pathogenic fungus that most frequently causes infections in humans. It is part of the microbiota commonly found in the skin, gastrointestinal tract, and vaginal mucosa. However, certain conditions, including immunosuppression, excessive use of antibiotics, hormonal changes, the use of medical devices in patients, and individual nutritional status, promote the development of opportunistic infections caused by this fungus. One of the main fungal structures interacting with the host is the cell wall, which is principally composed of chitin, glucan, and proteins. The cell wall plays key functions for the cell, such as osmotic protection; it is also responsible for cellular shape and acts as a signaling hub in response to environmental changes. Cell wall proteins participate in diverse cellular functions, such as attachment to surfaces and cell wall structure; some possess catalytic or transport activities. In this protocol, we show the methodology for isolating cell wall proteins covalently linked or not to cell wall components that can be previously labeled with [¹⁴C]-L-lysine by the action of the fungal transglutaminase localized in the cell wall. We use an extraction method by mechanical cell disruption and washing with 2 M NaCl, whose ionic strength eliminates contaminating proteins from other organelles, through subsequent serial treatments with SDS, chitinase, and zymolyase. Key features • Methodology for obtaining cell walls (CW) by mechanical cell disruption, fractionation, and isolation of CW proteins previously labeled with [14C]-L-lysine by the endogenous CW transglutaminase. • Extraction of CW noncovalently-linked proteins by sequential treatments with 2% SDS, and those covalently-linked CW proteins by chitinase and zymolyase treatment.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5456"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457841/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152284","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}