Bio-protocol最新文献

{"title":"Quantification of Total Free Radicals in <i>Drosophila</i> Using a Fluorescence-Based Biochemical Assay.","authors":"Shahira Helal Arzoo, Rubaia Tasmin, Surya Jyoti Banerjee","doi":"10.21769/BioProtoc.5238","DOIUrl":"10.21769/BioProtoc.5238","url":null,"abstract":"<p><p>Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), induce oxidative stress. This stress plays crucial roles in cellular signaling, stress response, and disease progression, making the quantification of free radicals essential for understanding oxidative stress mechanisms. Here, we present a high-throughput fluorescence-based protocol for measuring the presence of total free radicals, including ROS and RNS, in the whole adult <i>Drosophila melanogaster</i> (fruit fly). The protocol involves homogenizing whole adult flies in PBS and treating only the supernatant of the lysate with dichlorodihydrofluorescein-DiOxyQ (DCFH-DiOxyQ), which then converts into a fluorescent molecule, dichlorofluorescein (DCF), upon reacting with free radicals. The level of fluorescence is directly proportional to the amount of free radicals present in the sample. This protocol offers simplicity, scalability, and adaptability, making it ideal for studying oxidative stress in the model organism <i>Drosophila</i> and its different tissues under different dietary regimes, environmental stresses, genetic mutations, or pharmacological treatments. It is to be noted that the protocol uses a kit from Abcam, which has been used to measure free radicals in mice, rats, human blood, and cell lines. It can also be applied to biofluids, culture supernatants, and cell lysates, making it suitable for a wide range of sample types beyond whole organisms or tissues. However, due to our research focus and expertise, here we describe a detailed protocol to measure free radicals responsible for inducing oxidative stress only in fruit flies. Key features • Quantifies total free radicals including ROS and RNS levels in adult <i>Drosophila melanogaster</i> using a fluorescence-based approach for oxidative stress studies. • Suitable for high-throughput analysis with a 96-well black plate format, simultaneously enabling efficient handling of multiple samples and standards. • Adaptable to different experimental conditions, including diverse ROS-inducing treatments and mutations in <i>Drosophila</i>. • Offers detailed instructions for reagent preparation, sample homogenization, fluorescence measurement, normalization, and statistical analysis of data to ensure reproducibility and accuracy across research settings. Graphical overview <b>Schematic workflow of the assay.</b> Whole adult fruit flies are homogenized in PBS buffer and centrifuged. The clear supernatant is carefully transferred into new tubes for further treatment with different reagents, loaded into a clear-bottom black 96-well plate, and treated with another set of reagents. The plate is then incubated, and fluorescence is measured using the Agilent BioTek Synergy H1 plate reader.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5238"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896775/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627181","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":"Annotated Bioinformatic Pipelines for Phylogenomic Placement of Mitochondrial Genomes.","authors":"Jessica C Winn, Aletta E Bester-Van Der Merwe, Simo N Maduna","doi":"10.21769/BioProtoc.5232","DOIUrl":"10.21769/BioProtoc.5232","url":null,"abstract":"<p><p>The limited standards for the rigorous and objective use of mitochondrial genomes (mitogenomes) can lead to uncertainties regarding the phylogenetic relationships of taxa under varying evolutionary constraints. The mitogenome exhibits heterogeneity in base composition, and evolutionary rates may vary across different regions, which can cause empirical data to violate assumptions of the applied evolutionary models. Consequently, the unique evolutionary signatures of the dataset must be carefully evaluated before selecting an appropriate approach for phylogenomic inference. Here, we present the bioinformatic pipeline and code used to expand the mitogenome phylogeny of the order Carcharhiniformes (groundsharks), with a focus on houndsharks (Chondrichthyes: Triakidae). We present a rigorous approach for addressing difficult-to-resolve phylogenies, incorporating multi-species coalescent modelling (MSCM) to address gene/species tree discordance. The protocol describes carefully designed approaches for preparing alignments, partitioning datasets, assigning models of evolution, inferring phylogenies based on traditional site-homogenous concatenation approaches as well as under multispecies coalescent and site heterogenous models, and generating statistical data for comparison of different topological outcomes. The datasets required to run our analyses are available on GitHub and Dryad repositories. Key features • An extensive statistical framework to conduct model selection and data partitioning and tackle difficult-to-resolve phylogenies. • Instructions for generating statistical data for comparison of different topological outcomes. • Tips for selecting mitochondrial phylogenomic (mitophylogenomic) approaches to suit unique datasets. • Access to the scripts, data files, and pipelines used to enable replication of all analyses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5232"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896780/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627148","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":"An Activity-Based Proteomics with Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) for Identifying Target Proteases in <i>Arabidopsis</i> Apoplastic Fluid.","authors":"Sayaka Matsui, Yoshikatsu Matsubayashi","doi":"10.21769/BioProtoc.5226","DOIUrl":"10.21769/BioProtoc.5226","url":null,"abstract":"<p><p>Plant proteases participate in a wide variety of biological processes, including development, growth, and defense. To date, numerous proteases have been functionally identified through genetic studies. However, redundancy among certain proteases can obscure their roles, as single-gene loss-of-function mutants often exhibit no discernible phenotype, limiting identification through genetic approaches. Here, we describe an efficient system for the identification of target proteases that cleave specific substrates in the <i>Arabidopsis</i> apoplastic fluid. The method involves using <i>Arabidopsis</i>-submerged culture medium, which contains apoplastic proteases, followed by native two-dimensional electrophoresis. Gel fractionation and an in-gel peptide cleavage assay with a fluorescence-quenching peptide substrate are then used to detect specific proteolytic activity. The active fraction is then subjected to mass spectrometry-based proteomics to identify the protease of interest. This method allows for the efficient and comprehensive identification of proteases with specific substrate cleavage activities in the apoplast. Key features • Targets <i>Arabidopsis thaliana</i> secreted protease but may be applicable to other plant species and intracellular proteases if protease-enriched samples are available. • The protocol involves an in-gel peptide cleavage assay of native two-dimensional gels diced with SAINOME plates, using a fluorescence-quenching substrate. • Facilitates the efficient identification of proteases with the desired activity from the entire sample, without restricting the analysis to a specific class of proteases.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5226"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896777/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627110","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":"Differentiation, Maintenance, and Contraction Profiling of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.","authors":"Matthijs Snelders, Ingrid van der Pluijm, Jeroen Essers","doi":"10.21769/BioProtoc.5222","DOIUrl":"10.21769/BioProtoc.5222","url":null,"abstract":"<p><p>The development of patient-derived cardiac disease models has advanced rapidly due to the progress of human-induced pluripotent stem cell (hiPSC) technologies. Many protocols detail individual parts of the entire workflow, from handling hiPSCs and differentiating them into cardiomyocytes to live contraction imaging via widefield/phase-contrast and fluorescence microscopy. Here, we propose a streamlined protocol that guides users through hiPSC culture, differentiation, expansion, and functional imaging of hiPSC cardiomyocytes. First, hiPSC maintenance and handling procedures are outlined. Differentiation occurs over a two-week period, followed by selective expansion to increase the yield of hiPSC cardiomyocytes. Comprehensive characterization and quantification enable detailed contraction profiling of these cells. Designed to be low-cost, this protocol is suited for applications in drug discovery, screening, and clinical testing of patient-specific phenotypes. The addition of cardiomyocyte expansion and automated analysis distinguishes our protocol from current approaches. Key features • Protocol for the maintenance, differentiation, and expansion of hiPSC cardiomyocytes. • Detailed guidance for characterization and functional imaging of hiPSC cardiomyocytes. • Streamlined workflow integrating state-of-the-art protocols streamlined into a cost-effective approach. • A complete timeline from hiPSC culture to contraction imaging achievable in as little as three weeks.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5222"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896766/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627138","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":"Microbial Biofilm Detection and Differentiation by Dual Staining Using Maneval's Stain.","authors":"B Nirmala, Balram Ji Omar","doi":"10.21769/BioProtoc.5228","DOIUrl":"10.21769/BioProtoc.5228","url":null,"abstract":"<p><p>Microbial biofilms are structured communities of microorganisms embedded in a self-produced extracellular matrix, adhering to surfaces. These biofilms enhance bacterial resistance to antibiotics, immune responses, and environmental stress. Current microscopy techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and fluorescence microscopy, are commonly used to visualize and differentiate biofilms. However, their high cost and complexity often render them impractical. In contrast, simpler methods like crystal violet and Congo red staining are limited in distinguishing bacterial cells from the biofilm matrix. This study introduces a cost-effective, dual-staining method using Maneval's stain to visualize and differentiate microbial biofilms. It requires only basic equipment and minimal reagents, making it ideal for routine use in clinical diagnosis and microbial research. Key features • This dual-staining method differentiates bacterial cells, biofilm matrix, and capsules in a single stain. • This method applies to both bacterial and fungal biofilm. • This method requires no specialized training or equipment, with the entire process completed within 30-45 min. • Stained slides can be stored for extended periods (months) without degradation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5228"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896776/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627155","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":"Annotated Bioinformatic Pipelines for Genome Assembly and Annotation of Mitochondrial Genomes.","authors":"Jessica C Winn, Aletta E Bester-Van Der Merwe, Simo N Maduna","doi":"10.21769/BioProtoc.5231","DOIUrl":"10.21769/BioProtoc.5231","url":null,"abstract":"<p><p>Mitochondrial genomes (mitogenomes) display relatively rapid mutation rates, low sequence recombination, high copy numbers, and maternal inheritance patterns, rendering them valuable blueprints for mapping lineages, uncovering historical migration patterns, understanding intraspecific population dynamics, and investigating how environmental pressures shape traits underpinned by genetic variation. Here, we present the bioinformatic pipeline and code used to assemble and annotate the complete mitogenomes of five houndsharks (Chondrichthyes: Triakidae) and compare them to the mitogenomes of other closely related species. We demonstrate the value of a combined assembly approach for detecting deviations in mitogenome structure and describe how to select an assembly approach that best suits the sequencing data. The datasets required to run our analyses are available on the GitHub and Dryad repositories. Key features • Tips and code for conducting de novo, reference-based, and hybrid assembly. • Guide to detecting deviations in the structure of the mitochondrial genome. • Step-by-step guide to annotating and comparing the characteristics of mitochondrial genomes. • Access to the scripts, data files, and pipelines used to enable replication of all analyses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5231"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896769/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627112","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":"Procedure for Reliable and Long-Lasting Ex Vivo Recordings of Sciatic Nerve Activity in Mice.","authors":"Shani Berkowitz, Zehavit Goldberg, Amir Dori, Nicola Maggio, Efrat Shavit-Stein, Jérôme Joël Devaux","doi":"10.21769/BioProtoc.5233","DOIUrl":"10.21769/BioProtoc.5233","url":null,"abstract":"<p><p>Changes in neuronal conduction are common in disease states affecting peripheral nerves. These alterations can significantly impact nerve function and lead to sensorimotor disabilities. In vivo electromyography recording is a well-established electrophysiological method that has been used for decades to assess sensory and motor functions in the nervous system. Nerve studies are challenging to conduct in vivo in rodents, and the involvement of muscle activity makes it difficult to isolate and assess nerve function independently. This protocol provides a comprehensive guide for accurate ex vivo sciatic nerve dissection and handling from mice. It includes the creation of a three-compartment chamber and the establishment of electrophysiological protocols, which enable differential recordings and the analysis of compound action potentials from various nerve fibers. This setup allows researchers to study the specific effects of drugs and pathologies on nerves from a mechanistic perspective. The setup is a stand-alone apparatus that does not require the use of suction electrodes and the maintenance of negative pressure, which can affect the signal-to-noise ratio and recording stability. Key features • Sciatic nerve electrophysiology recordings from mice. • Allows for testing of disease model effects. • Ex vivo setup enables accurate pharmacological tests. • User-friendly software acquisition and analysis of compound action potential response.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5233"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896770/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627174","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 Sequential Derivatization for Gas Chromatography-[Orbitrap] Mass Spectrometry-based Metabolite Profiling of Human Blood-based Samples.","authors":"Akrem Jbebli, Kateřina Coufalíková, Moira Zanaboni, Manuela Bergna, Renzo Picenoni, Jana Klánová, Elliott J Price","doi":"10.21769/BioProtoc.5196","DOIUrl":"10.21769/BioProtoc.5196","url":null,"abstract":"<p><p>Many small molecules require derivatization to increase their volatility and to be amenable to gas chromatographic (GC) separation. Derivatization is usually time-consuming, and typical batch-wise procedures increase sample variability. Sequential automation of derivatization via robotic liquid handling enables the overlapping of sample preparation and analysis, maximizing time efficiency and minimizing variability. Herein, a protocol for the fully automated, two-stage derivatization of human blood-based samples in line with GC-[Orbitrap] mass spectrometry (MS)-based metabolomics is described. The protocol delivers a sample-to-sample runtime of 31 min, being suitable for better throughput routine metabolomic analysis. Key features • Direct and rapid methoximation on vial followed by silylation of metabolites in various blood matrices. • Measure ~40 samples per 24 h, identifying > 70 metabolites. • Quantitative reproducibility of routinely measured metabolites with coefficients of variation (CVs) < 30%. • Requires a Thermo Scientific^TM TriPlus^TM RSH (or comparable) autosampler equipped with incubator/agitator, cooled drawer, and automatic tool change (ATC) station equipped with liquid handling tools. Graphical overview Workflow for profiling metabolites in human blood using automated derivatization.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5196"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896771/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627075","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":"Integrated Co-extraction Protocol for Transcriptomic and H NMR Metabolomic Analysis of Multi-species Biofilms.","authors":"Anaïs Séguéla, Oriane Della-Negra, Roselyne Gautier, Jérôme Hamelin, Kim Milferstedt, Rémi Servien, Marie-Ange Teste, Cécile Canlet","doi":"10.21769/BioProtoc.5237","DOIUrl":"10.21769/BioProtoc.5237","url":null,"abstract":"<p><p>Capturing produced, consumed, or exchanged metabolites (<i>metabolomics</i>) and the result of gene expression (<i>transcriptomics</i>) require the extraction of metabolites and RNA. Multi-omics approaches and, notably, the combination of metabolomics and transcriptomic analyses are required for understanding the functional changes and adaptation of microorganisms to different physico-chemical and environmental conditions. A protocol was developed to extract total RNA and metabolites from less than 6 mg of a kind of phototrophic biofilm: oxygenic photogranules. These granules are aggregates of several hundred micrometers up to several millimeters. They harbor heterotrophic bacteria and phototrophs. After a common step for cell disruption by bead-beating, a part of the volume was recovered for RNA extraction, and the other half was used for the methanol- and dichloromethane-based extraction of metabolites. The solvents enabled the separation of two phases (aqueous and lipid) containing hydrophilic and lipophilic metabolites, respectively. The ¹H nuclear magnetic resonance (NMR) analysis of these extracts produced spectra that contained over a hundred signals with a signal-to-noise ratio higher than 10. The quality of the spectra enabled the identification of dozens of metabolites per sample. Total RNA was purified using a commercially available kit, yielding sufficient concentration and quality for metatranscriptomic analysis. This novel method enables the co-extraction of RNA and metabolites from the same sample, as opposed to the parallel extraction from two samples. Using the same sample for both extractions is particularly advantageous when working with inherently heterogeneous complex biofilm. In heterogeneous systems, differences between samples may be substantial. The co-extraction will enable a holistic analysis of the metabolomics and metatranscriptomics data generated, minimizing experimental biases, including technical variations and, notably, biological variability. As a result, it will ensure more robust multi-omics analyses, particularly by improving the correlation between metabolic changes and transcript modifications. Key features • Co-extraction of metabolites and total RNA from 6 mg of dry biomass of phototrophic biofilms, notably oxygenic photogranules. • Biphasic metabolome extraction for the characterization of hydrophilic and lipophilic metabolites using ¹H NMR. • Total RNA extraction with sufficient quality and quantity for analysis of (meta)transcriptome. Graphical overview Metabolomic and RNA co-extraction from oxygenic photogranules.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5237"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896772/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627140","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":"Colorimetric Determination of Tungsten and Molybdenum in Biological Samples.","authors":"Aaron P Donaghy, Gerrit J Schut, Nana Shao, Farris L Poole, Michael W W Adams","doi":"10.21769/BioProtoc.5195","DOIUrl":"10.21769/BioProtoc.5195","url":null,"abstract":"<p><p>Molybdenum (Mo) and tungsten (W) are elements that are utilized in biological systems. They are typically incorporated into the catalytic sites of enzymes coordinated to an organic pyranopterin cofactor; Mo may also be present in the form of a FeMo cofactor. While Mo is used by all branches of life, only a few microbes are able to utilize W. In order to study Mo- and W-dependent enzymes, it is important to be able to measure Mo and W in biological samples. Methods for determining Mo and W content in biological samples currently involve expensive and time-consuming processes like inductively coupled plasma mass spectrometry (ICP-MS) and chelation ion chromatography. There are less intensive colorimetric methods for measuring W in abiotic samples, but these have not been adapted to biological samples like cytosolic extracts and purified proteins. Herein, we developed a colorimetric assay based on the complexation of quercetin to molybdate (MoO₄ ^2-) or tungstate (WO₄ ^2-), the oxyanion forms of Mo and W that readily form in denatured biological samples. In the assay, the absorbance of quercetin is redshifted proportionally to the concentration of tungsten or molybdenum, which can be measured spectrophotometrically. This protocol provides a rapid method for screening biological samples for both Mo and W, although it does not distinguish between them. Key features • This protocol is adapted from the method developed by El-Sayed et al. [1] for analyzing abiotic samples. • The protocol is designed for a 96-well plate format and optimized for analyzing protein samples. • Can be used over the range of 1-20 μM W or Mo. • Allows for rapid and high throughput Mo and W determination in samples during protein purification. Graphical overview <b>Overview of colorimetric determination of tungsten in protein samples using quercetin.</b> Protein samples (200 μL) are transferred to microcentrifuge tubes along with 50 μL of 2.5× nitric acid mix and incubated at 65 °C overnight. The tubes are spun at 20,000× <i>g</i> for 20 min and 200 μL of the supernatant is transferred to a holding plate. 40 μL of each sample is transferred from the holding plate to four wells on an optical plate using a multichannel pipette. 160 μL of ethanol blank mix is added to one well per sample and 160 μL of quercetin mix is added to the remaining wells of each sample. The plate is sealed and shaken to mix each well. The seal is removed, and absorbances at 419 nm of each well are measured in a plate reader.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 5","pages":"e5195"},"PeriodicalIF":1.0,"publicationDate":"2025-03-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11896774/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143627078","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}