Current protocols最新文献

{"title":"Generation of Conformation-Specific Monoclonal Antibodies for Integral Membrane Proteins","authors":"Natalie Sheldon,&nbsp;Gunasekaran Dhandapani,&nbsp;Junhoe Kim,&nbsp;Cathy J. Spangler,&nbsp;Chengli Fang,&nbsp;Jumi Park,&nbsp;Prashant Rao,&nbsp;Eric Gouaux","doi":"10.1002/cpz1.70142","DOIUrl":"10.1002/cpz1.70142","url":null,"abstract":"<p>Antibodies and their antigen-binding fragments, including fragment antigen-binding domains (Fabs) and single-chain variable fragments (scFvs), are extraordinary tools in all fields of biology, particularly in neuroscience, where they have been utilized for imaging, detection, and quantification studies. Most antibodies bind to unstructured or linear epitopes. Conformation-specific antibodies, by contrast, bind to 3D epitopes, recognizing native conformations of the target antigen, and have proven highly useful in X-ray crystallography as crystallization chaperones and in cryo-electron microscopy as fiducial markers. Moreover, because conformation-specific antibodies recognize 3D shapes of the antigen, they often have exquisite specificity and are useful in immunofluorescence studies and in isolation of antigen from native tissues. Over the past decade, our group has devoted effort to developing murine monoclonal antibodies (mAbs) against important synaptic receptors, particularly ionotropic glutamate receptors (iGluRs) and their auxiliary proteins. We have developed reproducible methods for generating high-quality mAbs for structural, biochemical, and imaging studies. In this article, we show how to prepare proteoliposomes (PLs), carry out immunization and track the immune response, perform hybridoma generation, and analyze the specificity, cross-reactivity, and competition of mAb binding via enzyme-linked immunosorbent assay and fluorescence-detection size-exclusion chromatography. Our PL-based method produces high-affinity, conformation-specific antibodies targeting diverse synaptic membrane receptors in 4 months. Here, we describe the relevant protocols in detail and document the mAbs, Fabs, and scFvs that we have produced against iGluRs and their auxiliary subunits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation of conformation-specific antibodies for integral membrane proteins</p><p><b>Support Protocol 1</b>: Detection of conformational antibodies using ELISA</p><p><b>Basic Protocol 2</b>: Expression and purification of monoclonal antibodies and their derivatives</p><p><b>Support Protocol 2</b>: Concentration and clarification of insect cell supernatant for Fab purification</p><p><b>Support Protocol 3</b>: Measurement of ionotropic glutamate receptor binding kinetics using Octet BLI System</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70142","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144140489","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":"Exploring Strategies for the Development of Galleria mellonella Eggs: Incubation and Activation Methods in Laboratory Rearing","authors":"Paulo Henrique Fonseca do Carmo,&nbsp;Patrícia Michelle Nagai de Lima,&nbsp;Fabiana Alves de Souza Silva,&nbsp;Jaqueline Lemes Ribeiro,&nbsp;Kevin Kavanagh,&nbsp;Juliana Campos Junqueira,&nbsp;Maíra Terra Garcia","doi":"10.1002/cpz1.70153","DOIUrl":"10.1002/cpz1.70153","url":null,"abstract":"<p><i>Galleria mellonella</i> larvae are widely used for microbiological and toxicological studies due to their reproducibility of results, low costs, and ease of handling. Despite these advantages, the maintenance of homogeneous colonies of <i>G. mellonella</i> in laboratory rearing can face many challenges. We proposed a standardized protocol for the incubation of <i>G. mellonella</i> eggs, aiming to enhance larval development and provide a consistent and accessible experimental model for microbiological research. One Basic Protocol and two Alternate Protocols were established to simulate different conditions for maintaining and activating <i>G. mellonella</i> eggs. Basic Protocol, titled “Traditional group (TG),” follows our conventional protocol for egg activation with eggs being collected and stored at 27°C throughout the culture period; Alternate Protocol 1, titled “Immediate group (IG),” where eggs are stored after collection at 16°C for 120 days before being incubated at 27°C; and Alternate Protocol 2, titled “Gradual group (GG),” where the incubation temperature is gradually reduced from 27° to 16°C at a rate of 0.5°C per day, requiring 22 days to reach the target temperature. The eggs are then held at 16°C until day 76, after which the temperature is gradually increased back to 27°C at the same rate, for a total incubation time of 120 days. As a result, temperature fluctuations significantly delayed larval development in both IG and GG groups. Notably, larvae in the IG condition exhibited altered phenotypes, including abnormal pigmentation and a reduced ability to form cocoons. In contrast, despite the developmental delay, larvae in the GG condition displayed phenotypes comparable to those in the TG group. We propose that when conventional egg activation protocol (TG) is not feasible for laboratory rearing, gradual and controlled temperature changes (GG conditions) can serve as an effective alternative to prolonged egg development, as larvae from TG and GG exhibit comparable phenotype profiles. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Traditional group (TG)</p><p><b>Alternate Protocol 1</b>: Immediate group (IG)</p><p><b>Alternate Protocol 2</b>: Gradual group (GG)</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70153","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144125838","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":"Dual Labeling of Newly Synthesized Proteins and Existing Proteins Using CG-SLENP","authors":"Bingbing X. Li,&nbsp;Xiangshu Xiao","doi":"10.1002/cpz1.70146","DOIUrl":"10.1002/cpz1.70146","url":null,"abstract":"<p>This article describes a robust method for dual labeling of newly synthesized proteins and existing proteins. Assessing the properties of individual newly synthesized proteins from the bulk proteome is challenging due to difficulty in specifically isolating them. Previous methods such as non-natural amino acid labeling, isotope-labeled amino acid labeling, and puromycin labeling are effective for ensemble measurements. However, these existing methods do not support live-cell tracking or dynamic studies for a specific target protein. We designed a <span>c</span>hemical <span>g</span>enetics–based method for <span>s</span>elective <span>l</span>abeling of <span>e</span>xisting and <span>n</span>ewly synthesized <span>p</span>roteins (_CG-SLENP). Using nuclear lamin A (LA) tagged with a HaloTag (HaloTag-LA) as an exemplar protein and various Halo ligands, we describe _CG-SLENP for labeling existing proteins and newly synthesized proteins. This approach can label these proteins either one at a time or dually in the same live cell. This method holds great potential for broader applications to study any given protein of interest.©2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: _CG-SLENP labeling using clickable Halo ligands</p><p><b>Basic Protocol 2</b>: _CG-SLENP labeling using fluorescent Halo ligands</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144118061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Determination of Chlorophyll Concentration in In Vitro Peanut Leaves","authors":"Paola C. Faustinelli,&nbsp;Alicia N. Massa,&nbsp;Néstor W. Soria,&nbsp;Eliana López Colomba,&nbsp;Paola A. Suárez,&nbsp;Marshall C. Lamb","doi":"10.1002/cpz1.70150","DOIUrl":"10.1002/cpz1.70150","url":null,"abstract":"<p>The detection of chlorophyll is a convenient method for assessing gene editing efficiency when generating mutations in the <i>phytoene desaturase</i> (<i>PDS</i>) gene, which is related to chlorophyll biosynthesis. Various instruments and protocols are available for measuring chlorophyll; however, a minimum leaf area is required for precise quantifications. <i>In vitro</i> plant regeneration often requires several months to produce enough leaf tissue for chlorophyll content analysis. In this study, we optimized an existing chlorophyll quantification method by using a minimal amount of <i>in vitro</i> leaf tissue (&lt;2.0 mg) and a microvolume of chlorophyll extraction solution (3 µl) to quantify chlorophyll <i>a</i>, chlorophyll <i>b</i>, and total chlorophyll concentration using a UV-Vis spectrophotometer. This method will serve as a basic protocol for evaluating gene editing efficiency by correlating degrees of albinism (pigment content) with changes in the <i>PDS</i> gene sequence. Published 2025. This article is a U.S. Government work and is in the public domain in the USA.</p><p><b>Basic Protocol</b>: Determination of chlorophyll content in <i>in</i> <i>vitro</i> peanut leaves</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144118024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purification of Recombinant Histones and Mononucleosome Assembly","authors":"Yiming Zhao,&nbsp;Jingjun Hong","doi":"10.1002/cpz1.70155","DOIUrl":"10.1002/cpz1.70155","url":null,"abstract":"<p>The nucleosome is the basic unit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a <span>histone octamer</span>. Each histone octamer is composed of two copies each of the histone proteins <span>H2A</span>, <span>H2B</span>, <span>H3</span>, and <span>H4</span>. Nucleosomes play an important role in gene expression and regulation. While previous protocols use HPLC (high-performance liquid chromatography) to purify each histone and, after octamer reconstitution, subsequent FPLC (fast protein liquid chromatography) to purify the octamers. Here we present a method to carry out octamer reconstitution and mononucleosome assembly with FPLC only. This basic protocol describes a procedure for histone purification from <i>Escherichia coli (E. coli)</i>, their subsequent reconstitution as octamers, and assembly into mononucleosomes <i>in vitro</i>. Through this protocol, histone octamers and mononucleosomes can be reconstituted on a large scale without the use of HPLC. Overall, this protocol describes an effective new method for the reconstitution of octamers and assembly of mononucleosomes. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Purification of recombinant histones and mononucleosome assembly</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144118025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Visualization of the Cytoskeleton in Fixed Drosophila Embryos","authors":"Junnan Fang,&nbsp;Jovan S. Brockett,&nbsp;Weiyi Tian,&nbsp;Yuqing Hua,&nbsp;Elizabeth R. Gavis,&nbsp;Dorothy A. Lerit","doi":"10.1002/cpz1.70145","DOIUrl":"10.1002/cpz1.70145","url":null,"abstract":"<p>Embryogenesis necessitates the precise orchestration of cellular events to establish tissue patterning, developmental robustness, and viability. Syncytial embryogenesis, as in <i>Drosophila melanogaster</i>, poses added challenges as the synchronous and rapid nuclear divisions prior to cellularization occur within a shared cytoplasm. While the first several rounds of nuclear divisions occur within the center of the embryo, the nuclei progressively migrate peripherally, giving rise to the syncytial blastoderm. This spatial choreography hinges upon the dynamic interplay of actin and microtubules. Actin and microtubules coordinate nuclear division and position while preventing deleterious nuclear collisions. Additionally, the cytoskeleton also facilitates the segregation of organelles and molecular cargoes, including cell fate determinants required for cellular differentiation. As development progresses, actin and microtubules drive cellularization events for both germline and somatic cell lineages. Cytoskeletal disruption causes developmental arrest and embryonic lethality, underscoring its importance for embryogenesis. Given the significance of the cytoskeleton to these events, its visualization remains a cornerstone of cell and developmental biology research. Indeed, studies of the <i>Drosophila</i> embryo cytoskeleton have yielded valuable insights into cell biological mechanisms and developmental pathways conserved in various systems. Nevertheless, achieving optimal preservation of filamentous cytoskeletal structures poses technical challenges. Here, we present an embryo fixation method tailored to enhance the visualization of actin and microtubules via standard light microscopy approaches. This protocol complements immunofluorescence and molecular labeling techniques, including the direct labeling of fluorescently tagged proteins or mRNAs. By enabling detailed analysis of the cytoskeleton, this method expands opportunities to investigate the molecular mechanisms underlying embryo development and related processes. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of embryos for immunofluorescence of actin or microtubules</p><p><b>Basic Protocol 2</b>: Coupling immunofluorescence of the cytoskeleton with visualization of mRNAs via smFISH</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143950100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Navigating the Scholarly Literature: A Practical Guide to Searching Effectively (Without Too Much Stress)","authors":"Kathryn P. Kohn","doi":"10.1002/cpz1.70138","DOIUrl":"10.1002/cpz1.70138","url":null,"abstract":"<p>Effectively searching the scholarly literature is a fundamental academic skill. However, the process can be overwhelming due to the vast amount of available research and the complexity of academic databases. This overview article provides a practical guide to navigating the literature with confidence, outlining key strategies for identifying relevant sources, refining search queries, and troubleshooting common challenges. © 2025 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70138","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143930204","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":"Highly Effective Chemical Ligation of DNA and l-aTNA","authors":"Hikari Okita,&nbsp;Keiji Murayama,&nbsp;Hiroyuki Asanuma","doi":"10.1002/cpz1.70140","DOIUrl":"10.1002/cpz1.70140","url":null,"abstract":"<p>Chemical ligation of nucleic acids is a significant strategy for the establishment of a variety of functional biological tools and other applications. However, the conventional methodology has been suffering from low reaction efficiency or generation of unnatural structures at the ligation site. This article describes an effective chemical ligation method that connects the hydroxyl group and the monophosphate group at the nick site on the template, generating a natural phosphodiester bond. The method uses only <i>N</i>-cyanoimidazole (CNIm) and divalent metal cations to achieve chemical ligation in a template-directed manner, which can be applied to the ligations of not only DNA but also artificial nucleic acids such as <i>acyclic</i> <span>l</span>-threoninol nucleic acid (<span>l</span>-<i>a</i>TNA). Quantitative ligation is available within 10 min for <span>l</span>-<i>a</i>TNA fragments on an <span>l</span>-<i>a</i>TNA template, and within 2 hr for DNA fragments on a DNA template under optimized conditions. The effective chemical ligation system reported here will enable the development of biotechnology and nanotechnology, including exploration of <span>l</span>-<i>a</i>TNA aptamer via in vitro selection, chemical synthesis of genome-sized DNA and <span>l</span>-<i>a</i>TNA, functionalization of nanostructure, and creation of an <span>l</span>-<i>a</i>TNA-based artificial life in the future. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Chemical ligation using CNIm and divalent metal cation</p><p><b>Basic Protocol 2</b>: Imaging analysis of ligation reaction</p><p><b>Basic Protocol 3</b>: Mass spectrometry of ligation products</p><p><b>Support Protocol</b>: Kinetic analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143914533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Streaming Long-Read Sequence Alignments for HLA Predictions Using HLAminer","authors":"René L. Warren,&nbsp;Inanc Birol","doi":"10.1002/cpz1.70144","DOIUrl":"10.1002/cpz1.70144","url":null,"abstract":"<p><i>Current Protocols</i> is issuing corrections for the following protocol article.</p><p>Warren, R. L., &amp; Birol, I. (2025). Streaming long-read sequence alignments for HLA predictions using HLAminer. <i>Current Protocols</i>, <i>5</i>, e70124. doi: 10.1002/cpz1.70124</p><p>In the above-referenced article:</p><p>In Table 4, line 2, the dataset value has been changed from “NA12878, dataset A” to “NA19240, dataset A”.</p><p>The current version online now includes this correction and may be considered the authoritative version of record.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70144","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143909025","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":"Highly Adaptable Analysis Tools for Mapping Spatial Features of Cellular Aggregates in Tissues","authors":"Andrew Sawyer,&nbsp;Nick Weingaertner,&nbsp;Ellis Patrick,&nbsp;Carl G. Feng","doi":"10.1002/cpz1.70135","DOIUrl":"10.1002/cpz1.70135","url":null,"abstract":"<p>Multiplex imaging technologies have developed rapidly over the past decades. The advancement of multiplex imaging has been driven in part by the recognition that the spatial organization of cells can represent important prognostic biomarkers and that simply studying the composition of cells in diseased tissue is often insufficient. There remains a lack of tools that can perform spatial analysis at the level of cellular aggregates (a common histopathological presentation) such as tumors and granulomas, with most analysis packages focusing on smaller regions of interest and potentially missing patterns in the overall lesion structure and cellular distribution. Here, we present protocols to quantitatively describe the cellular structure of entire tissue lesions, built around two novel metrics. The Total Cell Preference Index reports whether a lesion tends to change in density in its central versus peripheral areas and can indicate the extent of necrosis across the entire lesion. The Immune Cell Preference Index then reports whether each immune cell type is located more centrally or peripherally across the entire lesion. The output of both indexes is a single number readout for simple interpretation and visualization, and these indexes can be applied to lesions of any size or shape. This simplifies cross-lesion comparison compared to traditional Euclidian distance–based analysis, which outputs multiple values for each lesion (one for output for each band used in the infiltration analysis). Additionally, this approach can be applied to any slide-scanning multiplexed imaging system, based on either protein or nucleic acid staining. Finally, the approach uses the open-source software QuPath and can be utilized by researchers with a basic understanding of QuPath, with the full analysis able to be applied to pre-generated images within 1 hr. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Image preparation and lesion selection</p><p><b>Basic Protocol 2</b>: Total Cell Preference Index and Immune Cell Preference Index</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 5","pages":""},"PeriodicalIF":2.2,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70135","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143904887","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}