Bio-protocol最新文献

{"title":"High-Throughput Indirect Monitoring of TORC1 Activation Using the pTOMAN-G Plasmid in Yeast.","authors":"Melissa Gómez, Guilherme Rocha, Diego Ruiz, Claudio Martínez, Francisco Salinas, Eduardo I Kessi-Pérez","doi":"10.21769/BioProtoc.5356","DOIUrl":"10.21769/BioProtoc.5356","url":null,"abstract":"<p><p>The target of rapamycin complex 1 (TORC1) is a highly conserved protein complex whose primary function is to link nutrient availability to cell growth in eukaryotes, particularly nitrogen sources. It was originally identified during the screening of <i>Saccharomyces cerevisiae</i> strains resistant to rapamycin treatment. For its part, <i>S. cerevisiae</i> is well known for being a key model organism in biological research and an essential microorganism for the fermentation of food and beverages. This yeast is widely distributed in nature, with domesticated and wild strains existing. However, little is known about what effects domestication has had on its different phenotypes; for example, how nitrogen sources are sensed for TORC1 activation and what impact domestication has had on TORC1 activation are questions that still have no complete answer. To study the genetic basis of TORC1 activation associated with domestication through approaches such as quantitative trait loci (QTL) mapping or genome-wide association studies (GWAS), and more generally for any study requiring TORC1 activity as a readout for a large number of individuals, it is necessary to have a high-throughput methodology that allows monitoring the activation of this pathway in numerous yeast strains. In this context, the present protocol was designed to assess phenotypical differences in TORC1 activation using a new reporter plasmid, the pTOMAN-G plasmid, specifically designed to monitor TORC1 activation. As a proof of concept, this methodology allowed phenotyping a large population of yeast strains derived from the <i>1002 Yeast Genomes Project</i>, the most complete catalog of genetic variation in yeasts. This protocol proved to be an efficient alternative to assess TORC1 pathway activation compared to techniques based on immunoblot detection, which, although effective, are considerably more laborious. Briefly, the protocol involves the design and construction of the pTOMAN-G plasmid, which carries a construct containing the firefly luciferase gene (<i>Luc</i>) under the control of the TORC1-regulated <i>RPL26A</i> gene promoter (<i>P<sub>RPL26A</sub></i> ). The protocol then details the process for selecting subgroups of yeasts based on their ability to grow under nutrient-limited conditions, using proline as the sole nitrogen source. These yeasts are then transformed with the TOMAN-G plasmid, using two alternative transformation methods. Finally, those yeasts that emit luminescence are selected, whose phenotype for TORC1 activation is measured by a nitrogen-upshift experiment in microculture. This approach, using the pTOMAN-G plasmid, offers a rapid and consistent method for assessing TORC1 signaling pathway activation in a large number of yeast strains, highlighting its usefulness to study the activation of the TORC1 pathway and the domestication process associated with it. In the future, a redesign of the plasmid could extend its use as a reporter tool to monitor the ac","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5356"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222639/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577228","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":"Optimized Midgut Tissue Dissociation of Mosquitoes and Sandflies for High-Quality Single-Cell RNA Sequencing.","authors":"Ana Beatriz F Barletta, Octavio Augusto Talyuli, Pedro Cecilio, Carolina Barillas-Mury","doi":"10.21769/BioProtoc.5352","DOIUrl":"10.21769/BioProtoc.5352","url":null,"abstract":"<p><p>Single-cell RNA sequencing has revolutionized molecular cell biology by enabling the identification of unique transcription profiles and cell transcription states within the same tissue. However, tissue dissociation presents a challenge for non-model organisms, as commercial kits are often incompatible, and current protocols rely on tissue enzymatic digestion for extended periods. Tissue digestion can alter cell transcription in response to temperature and the stress caused by enzymatic treatment. Here, we propose a protocol to stabilize RNA using a deep eutectic solvent (Vivophix, Rapid Labs) prior to tissue dissociation, thereby avoiding transcription changes induced by the process and preventing RNase activity during incubation. We validated this methodology for three medically important insect vectors: <i>Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis.</i> Single-cell RNA sequencing using our insect midgut dissociation protocol yielded high-quality sequencing results, with a high number of cells recovered, a low percentage of mitochondrial reads, and a low percentage of ambient RNA-two hallmark standards of cell quality. Key features • This protocol stabilizes tissue RNA before dissociation, avoiding RNase-mediated RNA degradation and transcription changes during the dissociation process. • Current protocols for insect midgut dissociation use enzymatic digestion of live tissues at temperatures that are incompatible with insect physiology. • We validated this new stabilization-dissociation methodology for insect midgut, yielding high-quality single-cell RNA sequencing with high gene counts, low mitochondrial RNA, and minimal ambient RNA contamination. Graphical overview Mosquito and sand fly midgut RNA stabilization and dissociation protocol for single-cell sequencing applications.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5352"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222636/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577230","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":"Protein Structural Characterization Using Electron Transfer Dissociation and Hydrogen Exchange-Mass Spectrometry.","authors":"Rupam Bhattacharjee, Jayant B Udgaonkar","doi":"10.21769/BioProtoc.5350","DOIUrl":"10.21769/BioProtoc.5350","url":null,"abstract":"<p><p>Intermediate states are often populated during the folding and unfolding reactions of a protein, and their detection is very challenging as they form transiently. Structural characterization of these short-lived intermediate species is difficult as it requires high-resolution methodologies. Hydrogen exchange-mass spectrometry (HX-MS) can identify and yield direct structural information on folding and unfolding intermediates, as well as information about the cooperativity of the folding or unfolding processes. The mass distributions of intact protein molecules are obtained first to determine their exchange pattern. Then, segment-specific structural information is obtained by analyzing the fragments of the protein. Enzymatic digestion is widely used with HX to determine the sequence-specific structural changes that occur to the protein during folding or unfolding. However, if a protein is an inhibitor of the protease, then alternative methodologies are required. Using electron transfer dissociation (ETD), it is possible to fragment the protein inside a mass spectrometer, and segment-specific structural changes occurring during the folding and unfolding process can be determined. In the case of HX-ETD-MS, protein molecules are first allowed to undergo HX, followed by their fragmentation. Deuterium retention in each fragment is measured. Very little, if any, scrambling of deuterium across fragments occurs during ETD-enabled fragmentation; hence, there is little scope for misinterpretation of the HX data. Key features • Analysis of intact protein data allows the identification of the intermediate states even in native and native-like conditions. • Precursor mass is determined from the intact protein analysis. • Fragments undergoing cooperative and non-cooperative transitions during protein unfolding can be distinguished from each other using HX-ETD-MS. • Analysis of the protein fragments that are obtained using ETD also enables the determination of the sequence of structural changes occurring in the protein. • HX-ETD-MS can provide structural insights into transiently formed intermediate states.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5350"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222632/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577232","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":"Activation of X-Succinate Synthases for Fumarate Hydroalkylation Using an In Vitro Activation Method.","authors":"Anshika Vats, Shukurah Anas, Ankush Chakraborty, Jian Liu, Jimyung Ryu, Sara M Mann, Mary C Andorfer","doi":"10.21769/BioProtoc.5357","DOIUrl":"10.21769/BioProtoc.5357","url":null,"abstract":"<p><p>X-succinate synthase enzymes (XSSs) are a class of glycyl radical enzymes (GREs) that play a pivotal role in microbial anaerobic hydrocarbon degradation. They catalyze the addition of hydrocarbons to fumarate using a protein-based glycyl radical, which must first be installed by a radical <i>S</i>-adenosylmethionine (rSAM) activating enzyme (AE). Once activated, XSS enzymes can undergo multiple catalytic cycles, forming C(sp³)-C(sp³) bonds with high stereoselectivity-a feature that highlights their potential as asymmetric biocatalysts. Due to the insolubility of XSS-AEs when heterologously expressed in <i>Escherichia coli</i>, studies have relied on in vivo radical installation protocols. Although these methods have illuminated fundamental details of XSS mechanisms, the inability to install a glycyl radical in vitro has limited biochemical studies and biotechnological advances using these enzymes. Here, we describe an in vitro protocol for reconstituting the activity of benzylsuccinate synthase (BSS), an XSS that catalyzes the addition of toluene to fumarate to form <i>R</i>-benzylsuccinate. To enable in vitro glycyl radical installation, we identified a soluble homolog via genome mining: 4-isopropylbenzylsuccinate synthase activating enzyme (IbsAE). IbsAE was expressed in <i>E. coli</i> and anaerobically purified in moderate yields (6-8 mg of protein per liter of culture); herein, we outline the expression and anaerobic purification of both IbsAE and BSS proteins. We describe a reproducible method for in vitro glycyl radical installation using these recombinant proteins and provide guidance on quantifying radical formation. Our optimized protocol consistently achieves 30%-50% radical installation, comparable to other in vitro GRE activations. Lastly, we demonstrate the application of this protocol for in vitro hydroalkylation reactions, achieving high assay yields (89%-97%). This protocol enables biochemical experiments that were previously challenging using cell extracts and accelerated advancements in XSS engineering and use in biocatalysis. Key features • Builds upon the method described by Andorfer et al. [1] to thoroughly describe in vitro activation and hydroalkylation using glycyl radical enzymes. • Useful for studying substrate scope and determining the stereoselectivity of XSS-catalyzed reactions with non-native substrates. • Can serve as a template for the reconstitution of activity for other XSS enzymes. • Describes protein production through hydroalkylation steps, which take approximately 6-7 days to complete, given that expressions and purifications are performed in parallel.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5357"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222630/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577224","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 Optimized RNA Extraction Method From Micro-quantities of Guinea Pig Cartilage and Synovium for Osteoarthritis Research.","authors":"Nidhi Bhardwaj, Diksha Rana, Jyotdeep Kaur","doi":"10.21769/BioProtoc.5348","DOIUrl":"10.21769/BioProtoc.5348","url":null,"abstract":"<p><p>Osteoarthritis (OA) is the primary cause of joint impairment, particularly in the knee. The prevalence of OA has significantly increased, with knee OA being a major contributor whose pathogenesis remains unknown. Articular cartilage and the synovium play critical roles in OA, but extracting high-quality RNA from these tissues is challenging because of the high extracellular matrix content and low cellularity. This study aimed to identify the most suitable RNA isolation method for obtaining high-quality RNA from microquantities of guinea pig cartilage and synovial tissues, a relevant model for idiopathic OA. We compared the traditional TRIzol^® method with modifications to spin column-based methods (TRIspin-TRIzol^®/RNeasy^TM, RNeasy^TM kit, RNAqueous^TM kit, and Quick-RNA^TM Miniprep Plus kit), and an optimized RNA isolation protocol was developed to increase RNA yield and purity. The procedure involved meticulous sample collection, specialized tissue processing, and measures to minimize RNA degradation. RNA quality was assessed via spectrophotometry and RT-qPCR. The results demonstrated that among the tested methods, the Quick-RNA^TM Miniprep Plus kit with proteinase K treatment yielded the highest RNA purity, with A_260:280 ratios ranging from 1.9 to 2.0 and A_260:230 ratios between 1.6 and 2.0, indicating minimal to no salt contamination and RNA concentrations up to 240 ng/μL from ⁓20 mg of tissue. The preparation, storage, homogenization process, and choice of RNA isolation method are all critical factors in obtaining high-purity RNA from guinea pig cartilage and synovial tissues. Our developed protocol significantly enhances RNA quality and purity from micro-quantities of tissue, making it particularly effective for RTqPCR in resource-limited settings. Further refinements can potentially increase RNA yield and purity, but this protocol facilitates accurate gene expression analyses, contributing to a better understanding of OA pathogenesis and the development of therapeutic strategies. Key features • Enables efficient RNA isolation from small, individual cartilage samples, eliminating the need for pooling and requiring minimal laboratory equipment. • Provides a reliable and cost-effective method for obtaining high-quality RNA suitable for RT-qPCR and gene expression analysis, from challenging tissue types like cartilage. Graphical overview Graphical representation comparing modified RNA isolation protocols for efficient RNA extraction from guinea pig cartilage and synovium.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5348"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222635/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577226","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 of Whey Protein Isolate-Based Microgel Targeted Delivery in Mouse Kidney.","authors":"Oksana A Mayorova, Mariia S Saveleva, Daria A Terentyeva, Olga I Gusliakova, Olga A Sindeeva","doi":"10.21769/BioProtoc.5347","DOIUrl":"10.21769/BioProtoc.5347","url":null,"abstract":"<p><p>Every year, there is an increase in the number of cases of chronic kidney disease, and a delay in the initiation of adequate treatment can lead to kidney failure, which requires regular dialysis or transplantation. Intensive systemic therapy used to treat kidney diseases often has a negative impact on other weakened organs, making it crucial to ensure targeted delivery of medications directly to the kidneys and to minimize systemic side effects. In order to reduce the toxicity of medications and decrease dosages, innovative delivery methods are being developed, such as micro-sized targeted delivery systems, which ensure highly effective distribution of encapsulated drugs directly within the organs. In a recent article, we presented innovative emulsified microgels stabilized with whey protein isolate (WPI), specifically designed for targeted drug delivery to the kidneys. Our stability studies revealed that these microgels start to degrade after 72 h, with this degradation exhibiting a time-dependent profile. Furthermore, intravenous administration of the microgel suspension through the tail vein showed significant selective accumulation in both the liver and kidneys over a duration of 5 days. As part of our research, we present the protocol for synthesizing emulsion microgels derived from whey protein isolate. This article provides a comprehensive overview of the procedures for precursor preparation, along with an in-depth investigation of the emulsion system's stability over time. The protocol also includes the injection of an emulsion microgel suspension into the tail vein of mice, enabling the evaluation of their biocompatibility and potential therapeutic efficacy. This protocol outlines the precautions and important nuances that should be considered at each stage of the experiment. Key features • This protocol provides detailed instructions on the synthesis of fluorescently labeled emulsion microgels using mucoadhesive whey protein isolate, allowing the replication of this process. • The protocol describes in detail how to prepare samples for confocal microscopy. • We are exploring a method to study the stability of emulsion microgels by analyzing the size of emulsion droplets using optical microscopy. • We provide guidance on the administration of suspension into the tail vein of mice, followed by visualization of the microgels' body biodistribution.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5347"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12229797/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585780","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":"Cloning-Free Targeting of Endogenous Loci to Generate Fluorescent Reporters in Medaka.","authors":"Simon Knoblich, Kiyoshi Naruse, Ali Seleit, Alexandre Paix","doi":"10.21769/BioProtoc.5360","DOIUrl":"10.21769/BioProtoc.5360","url":null,"abstract":"<p><p>CRISPR-Cas9 has democratized genome engineering due to its simplicity and efficacy. Adapted from a bacterial defense mechanism, CRISPR-Cas9 comprises the Cas9 endonuclease and a site-specific guide RNA. In vivo, the Cas9 ribonucleoprotein (RNP) can target specific genomic loci and generate double-strand breaks. Eukaryotic endogenous DNA repair mechanisms recognize the cut site and attempt to repair the DNA either by non-homologous end joining, which introduces insertions/deletions, resulting in a loss of reading frame in coding genes, or through homology-directed repair that maintains the reading frame. The latter approach allows the insertion of fluorescent reporter sequences in frame with protein-coding genes in order to monitor gene expression and protein dynamics in cells and whole organisms. Here, we provide a protocol for targeting endogenous genes to introduce sequences coding for fluorescent reporters in medaka (<i>Oryzias latipes</i>). The method is simple, robust, and efficient, thus facilitating straightforward organismal genome editing. Key features • Cloning free CRISPR/Cas9 tagging of endogenous genes with fluorescent reporter sequences. • Guidelines for designing CRISPR/Cas9 endogenous tagging experiments. • Straightforward generation of transgenic Medaka knock-in reporter lines. • Versatility with the use of Cas9 mRNA or protein and various fluorescent reporters.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5360"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222638/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577227","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":"The Establishment of 3D Polarity-Reversed Organoids From Human Endometrial Tissue as a Model for Infection-Induced Endometritis.","authors":"Xin Zhang, Li Zhang, Linyuan Fan, Zhaohui Liu","doi":"10.21769/BioProtoc.5349","DOIUrl":"10.21769/BioProtoc.5349","url":null,"abstract":"<p><p>Endometritis is a prevalent gynecological condition, often resulting from bacterial infections, which poses significant risks to women's reproductive health, including recurrent pregnancy loss, spontaneous abortion, and intrauterine adhesions. While conventional in vitro models have provided valuable insights into the pathogenesis of bacterial-induced endometritis, they often fail to replicate the complex cellular architecture and microenvironment of the endometrium due to species-specific differences and variations in the menstrual cycle. In this study, we present a novel organoid-based culture system that establishes a bacterial-induced endometritis model using endometrial organoids derived from primary epithelial cells. This protocol involves culturing endometrial organoids in a Matrigel-based three-dimensional matrix, followed by infection with <i>Escherichia coli</i> at a defined multiplicity of infection (MOI). The model effectively recapitulates key pathological features of bacterial-induced endometritis, including disruption of the epithelial barrier, release of inflammatory cytokines, and cellular damage. By preserving epithelial polarity, this approach offers enhanced physiological relevance, improves host-pathogen interaction studies, and provides a robust platform for evaluating potential therapeutic interventions. Key features • Establishes apical-out endometrial organoids to model pathogen-induced endometritis via natural infection routes. • Utilizes primary human endometrial epithelial cells to preserve cellular diversity and mimic the native endometrial microenvironment. • Provides a versatile platform for investigating host-pathogen interactions and evaluating potential therapeutic interventions in bacterial-induced endometritis. • Developed apical-out endometrial organoids to better mimic tissue structure and enhance pathogen infection for host-pathogen interaction studies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5349"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12222628/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144577233","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 Protein Purification Approach Using Elastin-Like Polypeptides (ELP) With His-Tag Assistance.","authors":"Young Kee Chae, Han Bin Shin","doi":"10.21769/BioProtoc.5342","DOIUrl":"10.21769/BioProtoc.5342","url":null,"abstract":"<p><p>Protein purification is essential for drug development, antibody production, and structural biology. We propose a cost-effective chromatography method using elastin-like polypeptide (ELP) as an aggregating core. In this approach, a chilled (target protein)-ELP fusion is loaded onto an immobilized metal affinity chromatography (IMAC) column equilibrated with low-salt buffer. Impurities are removed with warm high-salt buffer washes. Warming the column above the ELP's transition temperature (T_m) triggers ELP aggregation, physically trapping the target protein between beads. Subsequent stringent washing (high salt/imidazole) eliminates residual contaminants. Finally, cooling with cold low-salt buffer reverses aggregation, eluting the purified target protein. This method eliminates the need for advanced chromatography systems while achieving high purity through dual mechanisms: (1) IMAC affinity and (2) temperature-dependent physical capture. The ELP's reversible phase transition offers a simplified yet efficient purification platform, particularly valuable for lab-scale production of challenging proteins. Key features • This protocol requires an elastin-like polypeptide tag at the C-terminus of the target protein. • This protocol requires a His-Tag at the N-terminus of the target protein. • This protocol requires the use of colored/chromogenic proteins to enable real-time visual monitoring of chromatographic progression. • This protocol yields a highly pure protein by manually operating a Ni-bound resin at two different temperatures.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 12","pages":"e5342"},"PeriodicalIF":1.0,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254588/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144628075","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":"Single-Particle Tracking of AMPA Receptor-Containing Vesicles.","authors":"Victor C Wong, Deepika Walpita, Zhe J Liu, Erin K O'Shea","doi":"10.21769/BioProtoc.5325","DOIUrl":"10.21769/BioProtoc.5325","url":null,"abstract":"<p><p>AMPA-type receptors are transported large distances to support synaptic plasticity at distal dendritic locations. Studying the motion of AMPA receptor⁺ vesicles can improve our understanding of the mechanisms that underlie learning and memory. Nevertheless, technical challenges that prevent the visualization of AMPA receptor⁺ vesicles limit our ability to study how these vesicles are trafficked. Existing methods rely on the overexpression of fluorescent protein-tagged AMPA receptors from plasmids, resulting in a saturated signal that obscures vesicles. Photobleaching must be applied to detect individual AMPA receptor⁺ vesicles, which may eliminate important vesicle populations from analysis. Here, we present a protocol to study AMPA receptor⁺ vesicles that addresses these challenges by 1) tagging AMPA receptors expressed from native loci with HaloTag and 2) employing a block-and-chase strategy with Janelia Fluor-conjugated HaloTag ligand to achieve sparse AMPA receptor labeling that obviates the need for photobleaching. After timelapse imaging is performed, AMPA receptor⁺ vesicles can be identified during image analysis, and their motion can be characterized using a single-particle tracking pipeline. Key features • Track and characterize the motion of AMPAR GluA1⁺ vesicles in cultured rat hippocampal neurons. • GluA1 tagged with HaloTag (GluA1-HT) is expressed from native <i>Gria1</i> loci to avoid overexpression. • Sparse GluA1-HT labeling densities can be achieved without photobleaching via a block-and-chase strategy that utilizes Janelia Fluor (JF) dyes conjugated to HaloTag ligand (HTL). • GluA1-HT⁺ vesicles are identified during image analysis, and their motion is characterized using single-particle tracking (SPT) and hidden Markov modeling with Bayesian model selection (HMM-Bayes).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 11","pages":"e5325"},"PeriodicalIF":1.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12177357/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144334542","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}