Bio-protocol最新文献

{"title":"Fluorogenic Tissue-Based Assessment of Acid Ceramidase Activity.","authors":"Arielle Manabat, Debora Russo, Ilaria Penna, Rita Scarpelli, Laura Volpicelli-Daley","doi":"10.21769/BioProtoc.5778","DOIUrl":"10.21769/BioProtoc.5778","url":null,"abstract":"<p><p>Acid ceramidase (aCDase) is a lysosomal amidase that catalyzes the hydrolysis of sphingolipids (SphL), including ceramides and glucosylceramides. Altered expressions of aCDase are associated with several pathological conditions, such as cancer, inflammation, pain, and pulmonary disorders. aCDase activity is reduced in Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, diabetes, and cardiovascular disease. Recent reports suggest that aCDase inhibition may be an emerging strategy for treating several SphL-related neurodegenerative conditions, such as Krabbe, Gaucher, and Parkinson's disease, due to its role in the accumulation of glycosphingolipids. Therefore, the development of a tissue-based aCDase activity assay has potential applications in clinical diagnostics and drug discovery, enabling the evaluation of the onset and progression of disease from biological samples of patients, drug-target engagement analysis, and identification of biomarkers. Here, we report a detailed protocol for detecting aCDase activity in tissue lysates, using Rbm14-12 as a specific fluorogenic substrate for aCDase. Assay protocol optimization, including a procedure for the preparation and storage of tissue lysates and the identification of optimal protein tissue lysate amounts and substrate concentrations based on kinetic enzymatic parameter analyses, is described. Key features • This protocol relies on the use of the Rbm14-12 fluorogenic substrate. • This protocol was developed out of a need to measure the target engagement of an aCDase-targeting therapeutic in a Parkinson's disease-related animal model. • This protocol can be broadly useful for sensitively measuring aCDase activity in central and peripheral organ tissues.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5778"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507976/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835695","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 Step-by-Step Protocol for Efficient Global Accuracy Estimation of Protein Complex Structural Models with MViewEMA.","authors":"Lei Xie, Enjia Ye, Dong Liu, Guijun Zhang","doi":"10.21769/BioProtoc.5785","DOIUrl":"10.21769/BioProtoc.5785","url":null,"abstract":"<p><p>Estimation of model accuracy (EMA) is a critical step in protein structure prediction, enabling the ranking and selection of models in the absence of experimental structures. EMA methods aim to function independently of modeling approaches, ensuring broad applicability across diverse prediction workflows. Recent state-of-the-art EMA methods often improve estimation accuracy by incorporating consensus information from model pools, multiple sequence alignments (MSAs), structural templates, or protein language model representations. However, these strategies typically incur substantial computational cost or rely on information derived from the modeling process itself, which may introduce bias and compromise the independence of the assessment. This protocol describes the use of MViewEMA for global accuracy estimation of protein complex models from a single input structure. MViewEMA extracts residue-residue interaction features from complementary micro-, meso-, and macro-environmental perspectives and integrates multi-scale structural representations through a multi-view representation learning framework to predict global confidence scores. The protocol provides detailed procedures for input structure preparation, feature extraction, model inference, and global confidence score output, together with a tutorial for using the MViewEMA web server. The protocol provides a workflow based solely on structural information from the input model, achieving a balance between computational efficiency and estimation accuracy. It enables large-scale evaluation and selection of predicted models for protein structure prediction and downstream structural analysis applications. Key features • Provides an efficient and accurate single-model EMA framework for protein complex accuracy estimation. • Integrates residue-residue interaction features across micro-, meso-, and macro-environmental views through multi-view representation learning. • Performs independent protein structural model assessment without requiring MSAs, templates, protein language models, or consensus information from model ensembles. • Supports large-scale protein complex model ranking and selection for downstream structural analysis applications.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5785"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13589690/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149013353","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":"MORECOVERY: A Swab-Based Surface Sampling Protocol Incorporating a Nutrient-Free Resuscitation Step for the Detection of Clinically Relevant Gram-Negative Pathogens in the Viable but Non-Culturable State.","authors":"Daniela Visaggio, Cinzia Spagnoli, Massimiliano Lucidi, Marta Beccarini, Francesco Imperi, Paolo Visca","doi":"10.21769/BioProtoc.5782","DOIUrl":"10.21769/BioProtoc.5782","url":null,"abstract":"<p><p>Difficult-to-treat Gram-negative bacteria are a major cause of healthcare-associated infections due to multidrug resistance and limited therapeutic options. The hospital environment plays a central role in the persistence and transmission of infection, making environmental monitoring an essential component of infection prevention and control plans. Standard surface sampling techniques, including swabs, contact plates, and sponges, are widely used for environmental surveillance and are all based on culture-dependent methods. However, these techniques may underestimate the actual level of bacterial contamination since they fail to detect bacteria in the viable but non-culturable (VBNC) state, a reversible physiological condition in which bacterial cells remain viable but do not grow on conventional culture media. An innovative environmental sampling protocol, herein named MORECOVERY, has been developed to improve the detection of VBNC bacteria. The protocol integrates an essential resuscitation step, which improves the recovery of VBNC Gram-negative bacteria by a few orders of magnitude, into the standard swab-based sampling workflow. Following sample collection, swabs are incubated for 24 h at 37 °C in a carbon-free resuscitation buffer before plating, enabling the recovery of VBNC bacterial pathogens that would otherwise remain undetectable. By improving the recovery of VBNC cells, the MORECOVERY protocol allows a more accurate assessment of bacterial contamination of critical surfaces in healthcare settings. Its simplicity and minimal variation from standard workflows facilitate easy implementation in routine environmental monitoring. Key features • Enables the detection of Gram-negative bacteria in the viable but non-culturable (VBNC) state, allowing the recovery of otherwise undetectable bacterial contaminants. • Is compatible with standard swab-based environmental sampling workflows and routine microbiological procedures. • Relies on low-cost materials and standard laboratory equipment and can easily be implemented in healthcare facilities and other settings requiring careful monitoring of bacterial contamination. • Enhances the sensitivity of culture-based environmental surveillance methods through a simple resuscitation step prior to plating. • Combines a carbon-free resuscitation buffer and 24-h incubation at 37 °C, preventing bacterial growth while ensuring VBNC resuscitation without bacterial replication.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5782"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507968/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148834862","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":"Efficient and Fast Site-Directed Mutagenesis via Partially or Completely Overlapping Primer Pairs.","authors":"Paulina Varela-Castillo, Arezousadat Razavi, Changsheng Zhao, Martin M Geng, Amitis Nour, Xiang-Jiao Yang","doi":"10.21769/BioProtoc.5792","DOIUrl":"10.21769/BioProtoc.5792","url":null,"abstract":"<p><p>Site-directed mutagenesis is an indispensable molecular biology tool, but traditional methods often suffer from extended reaction time, structural limitations, and variable success rates. This article details three optimized protocols: P3a (primer pairs with 3'-overhangs, version a), P3b, and QuickChange 2.0, which rely on two highly processive DNA polymerases (Platinum SuperFi II and Q5) to accelerate and standardize plasmid engineering. The P3a method utilizes partially complementary primer pairs with distinct 3'-overhangs, achieving ~100% efficiency and enabling seamless cassette mutagenesis (insertion, deletion, and replacement). Building on this, the P3b method introduces specific thermal cycling modifications and a pre-denaturation step to overcome structural barriers resulting from GC-rich sequences. QuickChange 2.0 applies these two advanced polymerases to completely complementary primer pairs, even though the average efficiency decreases to 50%-60%. Replacing Pfu with the highly processive DNA polymerases also reduces PCR time to approximately 2 h. Thus, these new methods are more efficient and rapid than classical QuickChange mutagenesis based on Pfu polymerase. Key features • P3a uses 3'-overhang primers and superior polymerases for near-perfect efficiency in routine point mutations and seamless cassette mutagenesis. • P3b adapts the 3'-overhang primer design with specialized thermal cycling to successfully overcome structural barriers in highly GC-rich templates. • The QC2 protocol leverages completely complementary primers for fast and reliable introduction of point mutations and small insertions or deletions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5792"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507973/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835688","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":"Measurement of Net NH₄ ⁺ Fluxes Using the Non-invasive Micro-Test Technology (NMT) System in Rice.","authors":"Dong-Wei Di, Yunqi Liu, Bin Ye, Weiming Shi","doi":"10.21769/BioProtoc.5773","DOIUrl":"10.21769/BioProtoc.5773","url":null,"abstract":"<p><p>Ammonium (NH₄ ⁺) is the primary inorganic nitrogen source for rice (<i>Oryza sativa</i> L.). Substantial progress has been made in characterizing the functions of ammonium transporters (AMTs) in roots; however, the regulatory dynamics governing subcellular ammonium compartmentation after its entry into cells, particularly its vacuolar sequestration and efflux back to the external environment, remain poorly understood. This knowledge gap stems mainly from two factors: the difficulty of applying conventional detection methods at the organellar scale and interference caused by nonspecific ion adsorption to the cell wall of intact roots. To address these challenges, we present a detailed and reproducible protocol for real-time measurement of net NH₄ ⁺ fluxes in rice roots, root protoplasts, and isolated vacuoles using non-invasive micro-test technology (NMT). The protocol covers the preparation of protoplasts and vacuoles from rice roots, the configuration and calibration of the NMT system, and the step-by-step measurement of net NH₄ ⁺ fluxes at three distinct biological levels (intact roots, protoplasts, and vacuoles). By employing a unified sample preparation and measurement strategy, this protocol enables quantification of net uptake fluxes across the plasma membrane, characterization of net efflux dynamics under specific conditions, and indirect estimation of vacuolar sequestration capacity using the isolated vacuole system. Overall, this protocol provides a flexible and robust framework for studying NH₄ ⁺ homeostasis in plants and is readily adaptable to different crop species, treatment conditions, and experimental objectives. Owing to its modular design and compatibility with standard NMT equipment, it can be readily adopted by laboratories seeking to investigate nitrogen transport mechanisms in plants. Key features • Allows for testing of NH₄ ⁺ fluxes in roots, protoplasts, and vacuoles. • Applicable to plants grown under different culture systems, including <i>Arabidopsis thaliana</i> grown in dishes and rice grown in hydroponic systems. • Supports both long-term and transient stress treatments. • Real-time monitoring.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5773"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454944/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708927","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 Colorectal Tumor Spheroid Culture in Tunable Stiffness Alginate-Based Hydrogels and Subsequent Immunohistochemical Analysis.","authors":"Te Liu, Yantong Guo, Qi Wang, Yiyang Jia","doi":"10.21769/BioProtoc.5786","DOIUrl":"10.21769/BioProtoc.5786","url":null,"abstract":"<p><p>Tumor mechanical microenvironment, particularly extracellular matrix stiffness, plays a critical role in regulating cancer cell behavior, including proliferation, quiescence, and drug resistance. Conventional 2D culture or stiff 3D scaffolds fail to recapitulate the physiological soft (normal) or pathologically stiff (tumoral) mechanical niches. Here, we present a detailed protocol for establishing a tunable 3D tumor spheroid culture system using sodium alginate-based hydrogels crosslinked with calcium ions at different concentrations to achieve soft or stiff conditions that mimic normal colon and colorectal cancer tissues, respectively. We describe the step-by-step procedures for fabricating stiffness-tunable hydrogels, culturing colorectal cancer spheroids, releasing spheroids for downstream analysis, and performing immunohistochemical staining on intact spheroids. This protocol enables the reproducible investigation of mechanosensitive pathways and drug resistance mechanisms in a physiologically relevant 3D context. Key features • Tunable stiffness hydrogel system based on sodium alginate and calcium carbonate crosslinking. • 3D tumor spheroid culture that recapitulates normal and tumor mechanical microenvironments. • Gentle spheroid release using sodium citrate chelation, preserving morphology and viability. • Compatible with immunohistochemistry.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5786"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454884/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708938","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":"Mouse and Rat Oxygen-Induced Retinopathy Models to Study Vascular Features Seen in Retinopathy of Prematurity.","authors":"Morgan Paige Tankersley, Shreya Beri, Aniket Ramshekar, Bright Asare-Bediako, Neal Sandeep Shah, James Regun Karmoker, Heng-Chiao Huang, Elizabeth M Hartnett","doi":"10.21769/BioProtoc.5771","DOIUrl":"10.21769/BioProtoc.5771","url":null,"abstract":"<p><p>Retinopathy of prematurity (ROP), a retinovascular disease, is a leading cause of childhood blindness worldwide. Given the constraints of studying molecular mechanisms in preterm infants, reproducible animal models are important to understand ROP pathophysiology. Mouse and rat oxygen-induced retinopathy (OIR) models are the most commonly used and recapitulate key vascular features seen in ROP. However, these models are susceptible to inherent variability that limits reproducibility, including inter-litter variability, consistency of oxygen delivery across experiments, retinal dissection technique, and immunohistochemistry. Here, we describe a comprehensive protocol for performing the most common mouse and rat OIR models, and procedures such as eye enucleation, retinal dissection and flat mounting, isolectin GS-IB4 staining, whole retina stitched fluorescence imaging from Z-stacks, and quantification of vascular features. This protocol provides important materials and procedural details to increase the reproducibility of the mouse and rat OIR models. Key features • Rat and mouse oxygen-induced retinopathy (OIR) models. • Eye enucleation of experimental rat and mouse pups. • Retinal flat mounting and immunostaining for rat and mouse eyes. • Image analysis of retinal flat mounts from rat and mouse eyes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5771"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461755/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148726933","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":"Automated FLIM-FRET Segmentation Within RNP Condensates.","authors":"Noah D Powell, Joshua M Marcus, Leyla E Fahim, Jason E Lee","doi":"10.21769/BioProtoc.5775","DOIUrl":"10.21769/BioProtoc.5775","url":null,"abstract":"<p><p>Ribonucleoprotein (RNP) condensates are membraneless organelles that exist alongside many RNA-driven processes, such as transcription and splicing. Despite their ubiquity, the biological necessity of forming a condensed phase remains unclear, particularly because the same RNP components exist both within these organelles and in the surrounding dilute phase. Most current methods for studying biochemical interaction dynamics within condensates rely on in vitro reconstitution of minimal factors or low-throughput single-molecule studies. However, RNP condensates are complex organelles containing tens to hundreds of proteins and hundreds to thousands of different RNAs. Here, we describe a scalable, high-throughput fluorescence microscopy-based approach to analyze protein-protein interaction networks, allowing for the rigorous assessment of dynamic, process-critical interactions within RNP condensates from live cells. This method takes advantage of fluorescence lifetime imaging (FLIM) and phasor plot analysis to automate segmentation of condensate-localized fluorescence signals. Using suitable FLIM-Förster resonant energy transfer (FLIM-FRET) fluorescent pairs fused to proteins of interest, protein-protein interactions can be actively monitored throughout various conditions via changes in fluorescence lifetime. Results from this assay yield valuable insight into the organization and assembly of essential factors for different condensate-associated processes to infer the functional consequences of RNP granule partitioning. Although this protocol is tailored for studying protein interactions within condensates, the design and execution framework can be adapted to investigate protein-protein interactions across a wide variety of compartments within different biological systems. Key features • Automated segmentation of condensate-localized fluorescent signal independent of thresholding or background subtraction. • FLIM-FRET to analyze protein-protein interactions, with an emphasis on condensate interaction networks. • Monitor protein interactions from live cells over time and in response to different stimuli.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5775"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454946/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708960","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":"Coupled Enzyme Assay for Measuring Ornithine Decarboxylase Activity in Cell Lysates Using a Liquid-Stable CO₂ Detection Reagent.","authors":"Jung-Mao Hsu","doi":"10.21769/BioProtoc.5768","DOIUrl":"10.21769/BioProtoc.5768","url":null,"abstract":"<p><p>Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine biosynthesis that plays a critical role in cell proliferation and tumorigenesis. Reliable quantification of ODC activity is essential for mechanistic and therapeutic studies. Traditional assays often rely on radiolabeled substrates or discontinuous endpoint measurements. Here, we describe a non-radioactive, continuous spectrophotometric assay for measuring ODC activity in cell lysates using a commercially available liquid-stable CO₂ detection reagent. In this assay, CO₂ generated by ODC is captured as bicarbonate and utilized in a coupled enzymatic system containing phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH), leading to oxidation of thio-NADH. The decrease in absorbance at 405 nm due to thio-NADH oxidation is monitored in real time and is proportional to ODC activity. The protocol is performed in a 96-well plate format, requires minimal reagent preparation, and is suitable for medium- to high-throughput applications. Key features • Non-radioactive, continuous assay for measuring ODC activity. • Utilizes a commercially available liquid-stable CO₂ detection reagent, requiring minimal preparation and enabling improved reproducibility. • Real-time monitoring at 405 nm using a standard microplate reader. • Adaptable to a high-throughput 96-well format.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5768"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461751/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148726509","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 Novel Plate Reader-Based Protocol for Measurement of DNAJB6 Dimerization Activity.","authors":"Anna Gelman, Leif Kofoed Nielsen, Christian Hansen","doi":"10.21769/BioProtoc.5776","DOIUrl":"10.21769/BioProtoc.5776","url":null,"abstract":"<p><p>Progressive neurodegeneration linked to the accumulation of misfolded proteins is a hallmark of several neurodegenerative disorders, including Parkinson's disease, Huntington's disease, and Alzheimer's disease. Dysfunction in the protein homeostasis machinery correlates with pathology. The chaperone protein DNAJB6 is expressed in neurons and oligodendrocytes and has been shown to play a key role in preventing amyloid aggregation by binding to amyloidogenic proteins and facilitating their refolding or degradation, in cooperation with other chaperones. Here, we describe a simple and feasible assay that enables high-throughput screening for DNAJB6 activity in a plate reader format. We use genetically engineered HEK293 cells that stably express DNAJB6 fused to either CFP or YFP. These cells can be plated into multi-well plates, and the fluorescence resonance energy transfer (FRET) signal can be measured for analysis of DNAJB6 dimerization, which is linked to DNAJB6 activity. The protocol can be used for drug screening and to identify compounds that increase DNAJB6 dimerization, and can serve as a starting point for finding new medicines that act through modulating DNAJB6 activity. Key features • The protocol requires a plate reader capable of FRET analysis and bandwidth adjustment for CFP/YFP separation. It was developed using a CLARIOstar plate reader. • The protocol requires access to the authors' FRET DNAJB6 cell line or equivalent cells with stable expression of CFP/YFP-DNAJB6. • The assay measures DNAJB6 dimerization and can potentially be adapted to other proteins whose functional state is linked to dimerization activity. • The protocol is useful for compound screening purposes and requires pre-existing knowledge of basic cell culture.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5776"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454940/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708948","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}