Current protocols最新文献

Current Protocols is issuing a correction for the following protocol article.

Mueller, E. J., Rahimi, S., Sauta, P., Shojaeian, T., Durrer, L., Quinche, S., Francois, M., Locher, E., Edler, M., Illi, M., Gentinetta, T., Lau, K., Pojer, F., Borradori, L., & Hariton, W. V. J. (2024). Standardized production of anti-desmoglein 3 antibody AK23 for translational pemphigus vulgaris research. Current Protocols, 4, e1118. doi: 10.1002/cpz1.1118

In the above-referenced article:

The author name “Mueller” has been corrected to “Müller” throughout the article including the Biblography.

The current version online now includes this correction and may be considered the authoritative version of record.

This protocol presents methods for isolating and identifying novel nucleic acid–binding proteins. We focus on bacterial DNA-binding proteins, although the methods can be readily adapted to identify nucleic acid–binding proteins of eukaryotes or archaea, and proteins that bind to RNAs. Briefly, the DNA sequence of interest is affixed to beads and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute DNA-binding proteins that have greater specificity. Eluted proteins are then identified by standard proteomic techniques, such as mass spectrometry. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Lysis procedure

Basic Protocol 2: DNA affinity chromatography: Borrelia burgdorferi and Leptospira interrogans

Basic Protocol 3: Visualization and identification of DNA-binding proteins

We present a reproducible protocol for isolating and analyzing natural killer (NK) cells from freshly resected human glioblastoma multiforme (GBM) tissue using flow cytometry. The workflow yields viable single-cell suspensions suitable for downstream applications, including cell sorting, immunophenotyping, functional assays, and single-cell RNA-seq.© 2025 Wiley Periodicals LLC.

Basic Protocol 1: Dissociation of glioblastoma multiforme tissue to single-cell suspension

Support Protocol 1: Preparation of digestion dissociation mix for dissociation of glioblastoma multiforme tissue

Alternate Protocol 1: Mechanical-only dissociation of glioblastoma multiforme tissue (no-enzyme control)

Basic Protocol 2: Mononuclear cell enrichment and red blood cell lysis of glioblastoma multiforme single-cell suspension

Support Protocol 2: Removal of cellular debris from glioblastoma multiforme single-cell suspension

Basic Protocol 3: Flow cytometry staining and acquisition for identification of tumor-infiltrating NK cells from glioblastoma multiforme tissue

Alternate Protocol 2: Panel splitting for flow cytometry using instruments with limited numbers of channels

Current Protocols is issuing a correction for the following protocol article.

Zhao, Y., & Hong, J. (2025). Expression, purification, and ATP hydrolysis activity measurement of CtISWI and CtATPase. Current Protocols, 5, e70241. doi: 10.1002/cpz1.70241

In the above-referenced article:

Figure 14 has been replaced with the corrected version.

The current version online now includes this correction and may be considered the authoritative version of record.

Lathyrus sativus, commonly known as the grass pea, is a nutritious legume that is resilient to climate change, allowing it to grow in drought, waterlogged, and saline soils. However, developing effective functional genomic tools for this crop has been challenging, primarily due to the absence of reliable and stable transformation protocols. Agrobacterium rhizogenes-mediated hairy root transformation provides a practical and rapid method for validating gene functions using the CRISPR/Cas system. This method has not been applied to grass pea despite its potential. In this article, we present the first protocol for A. rhizogenes-mediated hairy root transformation and CRISPR/Cas genome editing aimed at the functional characterization of candidate genes in L. sativus. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Designing CRISPR/Cas9 construct for targeted gene editing in L. sativus

Support Protocol 1: Escherichia coli competent cell preparation and transformation

Support Protocol 2: A. rhizogenes competent cell preparation and transformation

Basic Protocol 2: A. rhizogenes-mediated hairy root transformation in L. sativus

Basic Protocol 3: Screening of transgenic hairy root lines

Support protocol 3: DNA isolation from L. sativus hairy roots

Chemotaxis is a fundamental biological process in which cells such as leukocytes migrate directionally from one site to another in response to a chemical gradient. This process of cellular migration is critical for execution of appropriate physiological responses to inflammation, as observed during immune defense and wound healing responses. Neutrophils being the first responders to the sites of infection or injury are one such type of cells. A neutrophil chemotaxis assay is a commonly used laboratory technique used to measure the directed movement of neutrophils toward a chemical signal termed a chemoattractant. It is used in immunology and inflammation research to study directed immune cell migration and to assess the effects of various molecules on cell migration. When studying neutrophil chemotaxis, current methods rely on counts of cells that have migrated across a membrane or substrate and observation of the migrated cells under a microscope. While useful, this approach fails to account for cells that have been activated but remain adherent to the substrate or cells that are in the intermediate stages of transmigration. Such limitations restrict the scope of information obtained and may lead to incomplete interpretations of neutrophil chemotaxis. To address this, we developed a new image analysis method that directly addresses the membrane in a chemotaxis chamber called the Boyden chamber. This improved technique allows quantification of neutrophils that have migrated as well as those that are adherent or actively transmigrating. By including all three stages of neutrophil movement, it provides a more comprehensive and physiologically relevant understanding of chemotaxis. It offers researchers a powerful tool to dissect the dynamics of immune cell behavior with greater sensitivity and accuracy, paving the way for deeper insights into immune responses and potential therapeutic interventions in inflammatory diseases. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Assay for the migration of bone marrow–derived neutrophils using the Boyden chamber

Basic Protocol 2: Analysis of adherent, transmigrating, and transmigrated cells

The chromatin immunoprecipitation followed by sequencing (ChIP-seq) assay is an instrumental and accurate method for understanding chromatin dynamics in eukaryotic cells. It provides critical insights into the regulation of gene expression and enables identification of regulatory elements, patterns of histone modifications, and chromatin states in health and disease conditions. Although cell cultures are great models to study molecular mechanisms associated with pathologies, studying tissues provides a physiologically native environment that reflects the cellular heterogeneity and spatial organization that are missing in an in vitro model. Several ChIP-seq protocols have been published; however, performing ChIP-seq in tissues remains a challenge in many settings due to the heterogeneity of tissues, complexity of cell matrices, low input material and intricacy of chromatin fragmentation and handling. Here, we present an optimized ChIP-seq protocol for solid tissues, with a focus on colorectal cancer. In this article, we incorporate simplified and efficient procedures for tissue preparation, chromatin extraction, immunoprecipitation, and library construction. The refined protocols overcome common limitations related to tissue processing and allows for highly reproducible, sensitive, and scalable analysis of disease-relevant chromatin states in vivo. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Frozen tissues preparation

Basic Protocol 2: Chromatin immunoprecipitation from tissues

Basic Protocol 3: Library construction for DNA sequencing

Basic Protocol 4: DNA nanoballs preparation for the DNBSEQ-G99RS sequencing platform and data quality control

Bulk RNA-sequencing (RNA-seq) is commonly used for identifying and characterizing genes through annotation, phylogeny, and differential expression analysis. For organisms without a reference genome, the need to generate a de novo transcriptome assembly for analyzing these data can prove challenging, as it is a multi-step process requiring many tools and computational resources. Therefore, a standardized and reproducible workflow using current best practices is required. We introduce the nf-core/denovotranscript workflow, an open-source solution built using Nextflow and the nf-core framework. The protocols here describe how the workflow can be used to perform pre-processing, de novo transcriptome assembly, redundancy reduction, assembly quality assessment, and quantification using RNA-seq data. This workflow offers simple installation and thorough documentation. The use of Docker, Singularity, and Podman container technologies also makes it portable across various computing environments. © 2025 Wiley Periodicals LLC.

Basic Protocol: Running nf-core/denovotranscript for de novo transcriptome assembly and quantification

Support Protocol: Installation and configuration for command line usage

This set of protocols was developed to detect Ascochyta rabiei, the fungus responsible for chickpea blight, in seeds. Given the significant impact of this disease and its potential to cause complete crop loss, even at low inoculum levels, optimizing detection protocols is crucial. This article proposes adjustments to key variables of a basic agar plate test—disinfection method, incubation, illumination, and culture medium—to improve microscopic identification (mainly using a stereomicroscope) and enhance differential growth of A. rabiei relative to other seed-borne fungi. Each step was carefully validated. Detection was verified through morphological and molecular confirmation of A. rabiei, as well as interlaboratory repeatability tests. In addition, the first distribution map of A. rabiei in Argentina was generated based on chickpea seed analyses conducted in the past decade and using the protocols outlined in this article. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Seed sampling procedure

Basic Protocol 2: Preparation of culture medium

Support Protocol: Sterilization of antibiotic

Basic Protocol 3: Seed disinfection by washing

Basic Protocol 4: Seed sowing and incubation

Basic Protocol 5: Detection of infected seeds

The cover image is based on the article Kinetic Analysis of HDAC- and Sirtuin-Mediated Deacylation on Chemically Defined Histones and Nucleosomes by Zhipeng Wang et al., https://doi.org/10.1002/cpz1.70251.