Bio-protocol最新文献

{"title":"Efficient Generation of Fetal Hepatic Stellate Cells From hiPSC.","authors":"Xia Yang, Hideki Taniguchi, Yun-Zhong Nie","doi":"10.21769/BioProtoc.5827","DOIUrl":"https://doi.org/10.21769/BioProtoc.5827","url":null,"abstract":"<p><p>Human induced pluripotent stem cell (hiPSC)-derived liver organoids have emerged as valuable models for studying human liver development. However, existing organoid systems often lack developmentally matched cell populations, particularly fetal hepatic stellate cells (HSCs), limiting their ability to recapitulate key developmental processes. Current approaches for generating HSCs rely on primary cells, immortalized cell lines, or hiPSC differentiation methods that frequently produce activated HSC-like cells and often require cell sorting. Here, we describe an efficient protocol for generating expandable fetal-like HSCs from hiPSCs through a stepwise differentiation strategy that mimics embryonic HSC development. The resulting cells can be robustly expanded while maintaining characteristic molecular and functional features of fetal HSCs. This protocol provides a reproducible and scalable source of fetal-like HSCs without cell sorting and supports the generation of multicellular liver organoids containing developmentally relevant stromal components. Beyond the validation of the protocol in studies of liver maturation and vascularization, it can be applied to investigations of HSC biology and congenital liver diseases. Key features • Efficient generation of fetal-like HSCs from hiPSCs without cell sorting. • Robust expansion of hiPSC-derived HSCs in vitro while maintaining fetal HSC characteristics. • Generation of developmentally relevant HSCs for multicellular liver organoid construction. • Applicable to studies of HSC biology, liver development, vascularization, and cell-cell interactions; successful implementation requires experience in hiPSC maintenance and differentiation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 19","pages":"e5827"},"PeriodicalIF":1.3,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13652712/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149169248","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 Accelerating Homozygous Line Screening in <i>Arabidopsis thaliana</i>.","authors":"Qiuyu Zhang, Yifan Wang, Chengqiao Jiang, Xueqin Zhou, Wenqing Zhong, Guanghui Yu","doi":"10.21769/BioProtoc.5819","DOIUrl":"10.21769/BioProtoc.5819","url":null,"abstract":"<p><p>Obtaining homozygous mutant and transgenic lines is a critical yet time-consuming step in <i>Arabidopsis thaliana</i> research. Conventional breeding procedures require seeds to undergo complete maturation and natural desiccation before harvest, followed by cold stratification to overcome seed dormancy. This process substantially prolongs generation turnover and delays genetic screening. Here, we describe a rapid germination strategy based on the use of partially dehydrated seeds collected approximately 15 days after pollination (DAP). At this developmental stage, embryos have reached physiological maturity, while the seeds have not yet entered deep desiccation-induced dormancy. After surface sterilization and short-term cold treatment (2-3 days at 4 °C), these seeds readily germinate on 1/2 MS medium and develop into normal seedlings. By bypassing the prolonged maturation and dormancy phases associated with naturally dried seeds, this protocol shortens each generation cycle by approximately 1-2 weeks. The method is particularly useful for accelerating the propagation of transgenic materials and the identification of homozygous mutant lines in <i>Arabidopsis</i>. Key features • The time-consuming and tedious screening of transgenic homozygous plants is a bottleneck limiting gene function research. • Provides a simple and effective approach for accelerating homozygous line identification in <i>Arabidopsis thaliana</i>. • Seeds harvested at 15 days after pollination can germinate normally, shortening the screening cycle by approximately 10 days per generation. • Applicable to T-DNA insertion mutants and transgenic lines carrying GFP or selectable markers, without requiring specialized equipment.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5819"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598652/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042334","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":"Isolation of Human Umbilical Cord Blood Hematopoietic Stem Cells and Directed Differentiation Into Megakaryocytes.","authors":"Wenjun Xia, Zeqing Miao, Weiwei Zhang, Zhixia Liu, Huang Wu, Yahan Fan","doi":"10.21769/BioProtoc.5822","DOIUrl":"10.21769/BioProtoc.5822","url":null,"abstract":"<p><p>Platelets originate from megakaryocytes, whose generation involves a series of biological processes including directed differentiation, proliferation, polyploidization, and maturation of hematopoietic stem cells. Abnormalities in megakaryocyte development and maturation can lead to quantitative and functional defects in platelets, thereby contributing to hemostatic or thrombotic disorders as well as the development of malignancies. Investigating megakaryocyte development and maturation and platelet production can provide important theoretical foundations for the diagnosis and treatment of thrombocytopenia, thrombotic diseases, and myeloproliferative neoplasms. Currently, there are three main clinical sources of hematopoietic stem cells (HSCs): bone marrow (BM), peripheral blood (PBSC), and umbilical cord blood (UCB). Among these, umbilical cord blood (UCB)-derived HSCs, due to their higher differentiation efficiency and stronger proliferative capacity, are the preferred starting cell source for studying megakaryocyte (MK) development and maturation and the mechanisms of platelet production. This article describes a detailed protocol covering all necessary steps for isolating CD34⁺ hematopoietic stem cells from umbilical cord blood, followed by in vitro induction culture with stem cell factor (SCF) and thrombopoietin (TPO) to generate mature megakaryocytes that highly express early megakaryocyte markers (CD41a, CD61) and late maturation markers (CD42a, CD42b). This protocol provides an effective tool for studying megakaryocyte development and platelet production and holds potential value for application in research on megakaryocyte-related diseases. Key features • Enables efficient isolation of highly pure and viable CD34⁺ hematopoietic stem cells from human umbilical cord blood using magnetic bead enrichment. • Establishes a serum-free, chemically defined culture system that minimizes batch-to-batch variability and ensures reproducible megakaryocyte differentiation. • Uses two key cytokines (SCF and TPO) to effectively induce directed differentiation of CD34⁺ cells into mature megakaryocytes. • This protocol is compatible with downstream analyses, including flow cytometry, qPCR, and platelet morphology, and can be used for disease modeling and drug screening.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5822"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598649/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042521","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":"Purification of MNase for Use in Ribosomal Profiling of High-Salinity Extremophiles.","authors":"Pavlina Gregorova, Matthew F Isada, Jocelyne DiRuggiero, Peter L Sarin","doi":"10.21769/BioProtoc.5811","DOIUrl":"10.21769/BioProtoc.5811","url":null,"abstract":"<p><p>Nucleases are key tools in molecular biology, enabling controlled nucleic acid digestion for applications such as ribosome profiling. Micrococcal nuclease (MNase) from <i>Staphylococcus aureus</i> is widely used as a tool in molecular biology and biochemistry, but its reduced activity under high-salt conditions necessitates higher enzyme input to achieve efficient digestion, increasing costs in studies of halophilic organisms. Here, we present an optimized protocol for the heterologous expression and purification of the recombinant staphylococcal MNase. The procedure enables reproducible production of a highly active, stable enzyme and incorporates an enzymatic activity assay to standardize batches to minimize variability. The resulting MNase exhibits robust activity in high-salt environments and remains stable during storage, providing a cost-effective and reliable alternative to commercial nucleases for ribosome profiling and related applications. Key features • Expression and one-step purification of MNase for use in ribosome profiling. • Validation of purified protein by absorbance-based enzymatic activity assay.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5811"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598657/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042689","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":"Homogeneous Time-Resolved Fluorescence-Based Assay to Screen ADP-Ribosyl Hydrolase Inhibitors.","authors":"Alice Yu, Duo Wu","doi":"10.21769/BioProtoc.5814","DOIUrl":"https://doi.org/10.21769/BioProtoc.5814","url":null,"abstract":"<p><p>ADP ribosylation (ADPr) is a crucial post-translational modification that plays a vital role in DNA damage repair. Catalyzed by ADP ribose polymerases using NAD⁺ as a substrate, ADPr activates DNA repair pathways rapidly, thereby maintaining genomic integrity. The involvement of ADP ribose hydrolases in this process is significant, as they hydrolyze PAR chains, facilitating the release of ADPr-modified proteins from DNA or other proteins, which is essential for subsequent DNA repair steps. This protocol outlines a high-throughput screening method for identifying inhibitors of ADP ribose hydrolases, utilizing His-Tb-conjugated and ADPr-modified His-ADP ribose polymerase as the signal donor, and GST-d2-conjugated GST-XRCC1 as the signal receptor. The detection of time-resolved fluorescence signals enables efficient evaluation of compounds with potential therapeutic activity against cancer. Key features • A high-throughput screening method is provided for efficiently identifying inhibitors of ADP ribose hydrolases. • The method employs homogeneous time-resolved fluorescence (HTRF) for sensitive detection of inhibitory effects on ADP ribose hydrolases, enabling high-throughput screening without complex separation steps. • The research aims to accelerate the development of novel cancer therapies targeting ADP ribose hydrolases to improve treatment outcomes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5814"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598656/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042514","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":"How to Perform a Tracer Displacement BRET Assay for the TRPML1 Ion Channel.","authors":"Micael R Cunha, Carolina M C Catta-Preta, Katlin B Massirer, Rafael M Couñago","doi":"10.21769/BioProtoc.5820","DOIUrl":"https://doi.org/10.21769/BioProtoc.5820","url":null,"abstract":"<p><p>The transient receptor mucolipin subtype 1 (TRPML1) is a ubiquitously expressed ion channel involved in lysosomal homeostasis. Recent pharmaceutical interest in developing agonist ligands has emerged due to beneficial effects in neurodegenerative diseases. The major high-throughput screening techniques to investigate this ion channel involve fluorescent calcium imaging and electrophysiology. Despite their high capacity for screening compounds, it is well known that both methods face hurdles, such as the need for expensive, specialized equipment. Here, we present a novel technique to screen for ligands of TRPML1 using a bioluminescence resonance energy transfer (BRET) assay. This assay consists of a target engagement assay in live cells, which permits the determination of binding constants between ligands and the target of interest in equilibrium or time-dependently. We employ a full-length TRPML1 C-terminally tagged with the small bioluminescent protein nanoluciferase. This ensures the correct localization of the ion channel in the lysosomal membrane and an optimal placement of the luciferase in the cytoplasm. We also developed a cell- and lysosome-permeable fluorescent BRET tracer that gives a BRET signal only when bound to the ion channel. This new protocol allows researchers worldwide to screen compounds that would interact with TRPML1 by using any plate reader with luminescent and fluorescence filters. Key features • This assay enables high-throughput screening of ligands for the TRPML1 ion channel without the need for a kinetic plate reader. • With the tracer displacement assay, it is possible to derive the apparent ligand affinity (K_dA) for TRPML1. • The assay uses full-length TRPML1, preserving the physiological context of lysosomal TRPML1.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5820"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598654/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042540","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 SPAAC-Based Bioorthogonal Method for Verifying Protein Palmitoylation.","authors":"Yaoyu Zhang, Jiahui Li, Xinxin Pei, Tengfei Ma, Yi Wang, Buchang Zhang, Changzhi Xu","doi":"10.21769/BioProtoc.5835","DOIUrl":"10.21769/BioProtoc.5835","url":null,"abstract":"<p><p>Palmitoylation is a crucial post-translational modification, and bioorthogonal chemistry based on azide-alkyne cycloaddition is typically used to verify protein palmitoylation. Traditional copper-catalyzed click chemistry (CuAAC) proceeds with fast kinetics and is widely used, but it requires a copper catalyst and suffers from copper-induced toxicity and nonspecific labeling. By contrast, strain-promoted click chemistry (SPAAC) has slower kinetics but is catalyst-free, offering high specificity, low cytotoxicity, and simple operation. However, SPAAC is mostly applied to live-cell labeling and imaging of known palmitoylated proteins in the field of palmitoylation, and its use in identifying novel palmitoylated proteins is still limited. Here, we present a SPAAC-based method for detecting endogenous protein palmitoylation. Compared with CuAAC, this method eliminates the need for copper catalysts and reducing agents, thereby simplifying the procedure and reducing reagent usage. Key features • A bioorthogonal method for identifying endogenous protein palmitoylation in cells. • Metabolical labeling of palmitoylated proteins in cells using azido palmitic acid. • The method requires simple steps and minimal reagents, reducing hands-on time and cost.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5835"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598653/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042288","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":"Digital Quantification of Membrane DAB Immunohistochemical Staining in FFPE Cervical Cancer Tissues Using an Open-Source CellProfiler Pipeline.","authors":"Kripa Krishnakumar, Nuha Rasheed, Nadira Nithyanandan, Roopak Murali, Leena Dennis Joseph, Ganesh Venkatraman, Rajesh Kumar Gandhirajan","doi":"10.21769/BioProtoc.5817","DOIUrl":"https://doi.org/10.21769/BioProtoc.5817","url":null,"abstract":"<p><p>Immunohistochemistry (IHC) is a highly specific and widely used laboratory technique for assessing protein localization and expression in tissue samples. Interpretation of 3,3' diamino benzidine (DAB)-based IHC is often based on observer-dependent manual scoring or traditional imaging software, which may show variability in DAB staining quantification. Furthermore, conventional image analysis tools often face limitations in precisely defining cell boundaries and quantifying membrane-specific signals. In this study, we present a standardized image analysis workflow using CellProfiler, an open-source software for image analysis for the quantification of membrane staining intensity in IHC images captured from slides prepared using formalin-fixed paraffin-embedded (FFPE) human cervical cancer tissue sections. The image analysis workflow was demonstrated using ASCT2 (SLC1A5), a membrane-localized amino acid transporter, as a representative biomarker for membrane-associated protein expression. This protocol involves image preprocessing, object identification, segmentation, and intensity measurement modules to distinguish cell membranes from cytoplasmic regions, enabling automated quantification of membrane intensity signals. The CellProfiler pipeline demonstrated improved accuracy in cell boundary identification and quantification of membrane-specific staining intensity. This is a rapid quantification process, since processing of each image only takes a few seconds; therefore, the analysis for 100 images can be performed within 10-15 min. This segmentation and quantification strategy is applicable to other membrane-based biomarkers after appropriate optimization of segmentation parameters. Following further minor modifications to the object identification modules, this pipeline can be used to detect and quantify cytoplasm- or nuclei-localized DAB-IHC markers across different tissue types. Overall, this protocol provides a standardized, user-friendly, and reproducible workflow for quantitative IHC image analysis that can be broadly applied to the study of protein biomarkers of different localizations, such as nuclei, cytoplasm, and cell membranes from different tissue types. Key features • Quantification of membrane-specific staining intensity in immunohistochemistry (IHC) images using a customized CellProfiler analysis pipeline. • Accurate delineation of cell boundaries, allowing separation of membrane and cytoplasmic signal regions. • Reproducible stepwise workflow adaptable to multiple tissue types and membrane protein biomarkers. • Applicability to studies of membrane-localized biomarkers, such as ASCT2, in cancer research.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5817"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598660/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042511","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":"Protecting Against Cytoplasmic Protein Aggregates With Cytoplasmic PML Variants.","authors":"Jia-Xin Zhu, Yuchen Xia, Jiaqi Liu, Li Cao, Steven X Hou, Yang Wang","doi":"10.21769/BioProtoc.5815","DOIUrl":"10.21769/BioProtoc.5815","url":null,"abstract":"<p><p>Cytoplasmic protein aggregation is a defining feature of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, and certain forms of motor neuron disease. Recent evidence indicates that promyelocytic leukemia protein (PML) and engineered PML-derived variants can act as versatile aggregate-remodeling factors. In particular, cytoplasmically redirected PML variants recognize pathological cytoplasmic inclusions and promote their clearance. Here, we describe a protocol to generate and validate two engineered cytoplasmic PML variants: full-length mPML, which is redirected to the cytoplasm by disruption of its nuclear localization sequence, and the truncated mPML^ΔRBC variant, which lacks the RING, B-box, and coiled-coil domain but retains aggregate-reducing activity. The protocol integrates fluorescence-based imaging, bimolecular fluorescence complementation, detergent-soluble/insoluble fractionation, and validation in primary rat cortical neurons. This workflow provides a practical platform for assessing cytoplasmic aggregate burden and for comparing the aggregate-remodeling activities of PML-derived constructs. It can also be adapted to other disease-associated aggregation-prone proteins, including TDP-43, SOD1, FUS, tau, polyGA, and polyQ-expanded proteins. Key features • Describes the generation and validation of two complementary cytoplasmic PML variants: full-length mPML and truncated mPML^ΔRBC. • Provides fluorescence-based and BiFC-based assays to visualize pathological cytoplasmic protein assemblies. • Includes detergent-soluble/insoluble fractionation for biochemical assessment of aggregate burden. • Establishes a primary rat cortical neuron workflow to evaluate the effect of mPML on TDP-43-CTF aggregates. • Can be adapted to other aggregation-prone proteins and additional PML-derived aggregate-remodeling candidates.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5815"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598655/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042445","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":"Proximity Labeling in <i>Caenorhabditis elegans</i> to Detect Neuronal Proteins During Memory Formation.","authors":"Aelon Rahmani, Yee Lian Chew","doi":"10.21769/BioProtoc.5821","DOIUrl":"10.21769/BioProtoc.5821","url":null,"abstract":"<p><p>Memory is a fundamental process, regulated by protein-protein interactions within neuronal proteome networks. Learning-dependent changes in specific brain regions important for memory have been detected by mass spectrometry, by comparing proteins from animals trained to learn with mock-trained controls. Detection through this method relies on relative protein abundance; brain dissection is readily available for macroscopic animals to spatially control protein identification by mass spectrometry. In the nematode <i>C. elegans</i>, however, such spatial control is limited due to its microscopic size, hindering its utilization in proteomics. A protocol to address this limitation would strengthen an already excellent model to study memory, given that many proteins for learning are evolutionarily conserved in the worm and single-cell expression is uniquely defined across all 302 neurons. We modified existing protocols to enable (i) proximity labeling detection of neuronal proteins in <i>C. elegans</i> and (ii) high-throughput enrichment of these proteins from >3,000 whole worm bodies simultaneously, to assess trained vs. mock-trained proteomes. This involved the biotin ligase enzyme TurboID, which promiscuously labels nearby proteins with its substrate biotin. Enzyme expression was transgenically restricted to the nervous system, and biotin supplementation was limited to the training (or mock training) period in a classical (gustatory) conditioning paradigm. Labeled proteins were enriched by pull-down using streptavidin, which has a high binding affinity to biotin, and then processed for mass spectrometry runs and qualitative data analysis. This protocol is uniquely advantageous in that it minimizes proteins present before a temporal window of interest (training/mock training), improving the detection of lowly abundant proteins from a specific tissue in the worm (neurons). We have demonstrated that the protocol can sufficiently detect novel learning regulators, thus providing a useful framework to interrogate proteomes in microscopic brains. Key features • This protocol assumes that users have fundamental knowledge about <i>C. elegans</i>. • The entire workflow typically takes approximately three weeks, with many opportunities to pause and revisit the procedure. • This protocol can be adapted for use in other animals and applied to a diverse array of temporally defined biological processes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 18","pages":"e5821"},"PeriodicalIF":1.3,"publicationDate":"2026-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13598659/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149042578","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}