Biology Methods & Protocols(ISSN: 2396-8923) publishes methodology and protocol papers in the biological sciences. It primarily publishes in the areas of genetics and heredity, biochemistry, molecular biology, ecology, evolutionary biology, marine and freshwater biology and cell biology.
Cold Spring Harbor Protocols is an interdisciplinary journal providing a definitive source of research methods in cell, developmental and molecular biology, genetics, bioinformatics, protein science, computational biology, immunology, neuroscience and imaging. Each monthly issue details multiple essential methods—a mix of cutting-edge and well-established techniques. All protocols are up-to-date and presented in a consistent, easy-to-follow format.
The Drosophila melanogaster neuromuscular junction (NMJ) is a superb system for studying synapse function. Beyond that, the NMJ is also great for studying forms of synaptic plasticity. Over the last 25 years, Drosophila NMJ neuroscientists have pioneered understanding of a form of plasticity called homeostatic synaptic plasticity, which imparts functional stability on synaptic connections. The reason is straightforward: The NMJ has a robust capacity for stability. Moreover, many strategies that the NMJ uses to maintain appropriate levels of function are mirrored at other metazoan synapses. Here, we introduce core approaches that neurophysiologists use to study homeostatic synaptic plasticity at the peripheral Drosophila NMJ. We focus on methods to study a specific form of homeostatic plasticity termed presynaptic homeostatic potentiation (PHP), which is the most well-characterized one. Other forms such as presynaptic homeostatic depression and developmental forms of homeostasis are briefly discussed. Finally, we share lists of several dozen factors and conditions known to influence the execution of PHP.
The Drosophila melanogaster neuromuscular junction (NMJ) is an easily accessible synapse and an excellent model for understanding synapse development, function, and plasticity. A form of plasticity called presynaptic homeostatic potentiation (PHP) operates at the NMJ and keeps synapse excitation levels stable. PHP can be induced rapidly in 10 min by application of a pharmacological antagonist of glutamate receptors (philanthotoxin-433) or chronically by deletion of the gene encoding the postsynaptic glutamate receptor subunit GluRIIA. To assess PHP, electrophysiological recordings of spontaneous miniature excitatory postsynaptic potentials and evoked excitatory postsynaptic potentials are usually performed at the NMJ of muscle 6 at abdominal segments A2 and A3. This protocol describes steps for larval dissection to access the NMJ, use of mutant lines to assess PHP, application of philanthotoxin-433 to the NMJ, and electrophysiological recordings following drug application. Collectively, these steps allow for analysis of the acute induction and expression of PHP. Recording chamber preparation, electrophysiology rig setup, larval dissection, and current clamp recording steps have been described elsewhere.
Presynaptic homeostatic potentiation (PHP) is a type of homeostatic regulation that stabilizes synaptic output under conditions where postsynaptic receptor function is impaired. PHP manifests as a significant increase in presynaptic neurotransmitter release, compensating for decreased postsynaptic receptor activity and thus maintaining stable excitation levels in postsynaptic cells. Presynaptic neurotransmitter release is calcium-dependent, initiated by calcium influx through voltage-gated calcium channels localized at the presynaptic active zones. This calcium influx triggers the fusion of vesicles from the readily releasable vesicle pool (RRP) that are ready for immediate release. Two key presynaptic cellular mechanisms are essential for PHP's induction and maintenance. First, a compensatory rise in the abundance of presynaptic calcium channels (and consequently, an increase in calcium influx) occurs when postsynaptic glutamate receptors are suppressed. Second, the RRP size enlarges during PHP. PHP is disrupted if either of these processes is impaired. This protocol outlines the use of the two-electrode voltage-clamp technique for assessing the RRP during PHP, induced either pharmacologically or genetically, at the Drosophila neuromuscular junction (NMJ). Electrophysiological recordings typically take place at the NMJ of muscle 6 in abdominal segments A2 and A3.
Synaptic transmission plays a critical role in information processing and storage within the nervous system. The triggering of action potentials activates voltage-gated calcium channels at presynaptic active zones, facilitating the calcium-dependent release of synaptic vesicles. Homeostatic mechanisms are crucial in stabilizing synaptic function. At the Drosophila neuromuscular junction, a compensatory increase in presynaptic neurotransmitter release occurs when postsynaptic glutamate receptor function is pharmacologically or genetically impaired, thereby stabilizing synaptic output. This adaptation is known as presynaptic homeostatic potentiation (PHP). Recent advancements, including confocal and super-resolution imaging techniques, have demonstrated an increase in presynaptic calcium influx during both the rapid induction and long-term maintenance of PHP. These observations indicate that the abundance and structural organization of presynaptic calcium channels, along with various active zone components, undergo modifications following the suppression of postsynaptic glutamate receptors. Such findings underscore the critical roles of trafficking and stabilization of presynaptic calcium channels and active zone proteins in homeostatic plasticity. This protocol describes using calcium indicators and confocal imaging methods to measure single-action potential-evoked presynaptic calcium influx during PHP.
Flavonoids represent a large class of phenolic specialized metabolites and play crucial roles in plant–environment interactions, including responses to biotic and abiotic factors. While the core flavonoid biosynthesis pathway is well known in several plant species, enzymes involved in modifying core flavonoid structures, furnishing them with distinct biological activities, continue to be identified. Anthocyanins, a specific type of flavonoid pigment, serve various functions, including attracting pollinators and seed-dispersing organisms when accumulated in flowers and seeds. Anthocyanins also accumulate in vegetative tissues of many plants, especially under unfavorable conditions. In this review, we present an overview of the diverse structures, various distributions, and multiple functions of flavonoids in plants.
Plants accumulate hundreds of thousands of specialized metabolites that participate in their interactions with the environment. Among these compounds, phenolics represent a large class, and they play important physiological roles, such as providing a first barrier against pathogens, cues to pollinators, and radiation protection. Maize is one of the most important crops worldwide for food, animal feed, and biofuels, and it has the potential to accumulate different phenolics in vegetative tissues as well as in seeds. Recent studies have identified a large number of phenolic compounds—with a diversity of chemical decorations—in different maize tissues, but these likely represent just a fraction of the metabolic diversity of maize. In this protocol, we describe a specific method for the extraction and quantification of maize phenolic compounds by ultra-high-pressure liquid chromatography–tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM-MS/MS) analysis. We provide detailed instructions for the extraction of phenolics using acidic methanol, and for the quantification of 33 different compounds in maize stems, including flavonoids, phenolic acids, and lignin precursors.
Anthocyanins are flavonoid pigments that accumulate in fruits and flowers that serve as attractants for pollinators and seed-dispersing organisms. Anthocyanins exhibit diverse chemical structures, characterized both by different anthocyanidin core structures and numerous chemical modifications of the anthocyanidin core. Here, we describe a protocol for the extraction and quantification of total anthocyanins, as well as for the characterization of anthocyanidin core structures and specific anthocyanins, using a spectrophotometer, high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography–two-dimensional mass spectrometry (UHPLC-MS/MS). The method involves anthocyanin extraction using acidic methanol, anthocyanin quantification using a spectrophotometer, determination of anthocyanidin core structure from hydrolyzed anthocyanin extracts using UHPLC-MS/MS, separation of different anthocyanins using HPLC, and characterization of specific anthocyanins using UHPLC-MS/MS. As an example, we describe how we have used this protocol to extract and quantify total anthocyanins from maize leaves, identify cyanidin as the core anthocyanidin structure, and characterize three specific anthocyanins that accumulate in maize leaves, each having a cyanidin core with decorations of a hexose group, and a malonyl or coumaroyl moiety.
Current Protocols in Chemical Biology (2160-4762) provides clear step-by-step instructions for validated protocols as well as enlightening overviews for researchers who need specialized chemical techniques and tools for their studies of biology and drug design
Cover: Reversible addition-fragmentation chain-transfer (RAFT) polymerization is a controlled radical polymerization process that generates synthetic polymers with controllable molecular weight with low dispersity These RAFT polymers can be conveniently tuned with a versatility that matches the broad needs of materials science including carriers for therapeutic and artificial extracellular matrices for tissue engineering. See Jiang et al. (http://doi.org/10.1002/cpch.85).
The emergence of covalent inhibitors and chemoproteomic probes in translational chemical biology research requires the development of robust biophysical and analytical methods to characterize their complex interactions with target biomolecules. Importantly, these methods must efficiently assess target selectivity and accurately discern noncovalent binding from the formation of resultant covalent adducts. One recently reported covalent chemical tool used in tumor immune oncology, covalent immune recruiters (CIRs), increases the proximity of immune cells and cancer cells, promoting immune recognition and response. Herein we describe biolayer interferometry (BLI) biosensor, flow cytometry, and solution fluorescence-based assay approaches to characterize CIR:antibody binding and CIR-antibody covalent-labeling kinetics. BLI technology, akin to surface plasmon resonance, provides the unique opportunity to investigate molecular binding and labeling kinetics both on a solid surface (Basic Protocol 1) and in solution (Alternate Protocol 1). Here, recruitment of mass-containing proteins to the BLI probe via CIR is measured with high sensitivity and is used as a readout of CIR labeling activity. Further, CIR technology is used to label antibodies with a fluorescent handle. In this system, labeling is monitored via SDS-PAGE with a fluorescence gel imager, where increased fluorescence intensity of a sample reflects increased labeling (Basic Protocol 2). Analysis of CIR:antibody target-specific immune activation is demonstrated with a flow cytometry‒based antibody-dependent cellular phagocytosis (ADCP) assay (Basic Protocol 3). This ADCP protocol may be further used to discern CIR:antibody binding from covalent adduct formation (Alternate Protocol 3). For the protocols described, each method may be used to analyze characteristics of any covalent-tagging or antibody-recruiting small molecule or protein-based technology. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Determining “on-probe” reaction kinetics of CIR1/CIR4 via biolayer interferometry with Octet RED96
Alternate Protocol 1: Determining “in-solution” reaction kinetics of prostate-specific membrane antigen targeting CIR (CIR3) via biolayer interferometry with Octet RED96
Basic Protocol 2: Reaction kinetics of covalently labeled antibodies via fluorescence SDS-PAGE
Basic Protocol 3: Small molecule‒directed antibody-dependent cellular phagocytosis on live human cells measured via flow cytometry
Alternate Protocol 2: Kinetic analysis of CIR3:antibody labeling via antibody-dependent cellular phagocytosis on flow cytometry
Support Protocol 1: Activation of U937 monocytes with interferon γ
Support Protocol 2: Labeling streptavidin beads with biotinylated prostate-specific membrane antigen receptor
Drug-induced liver injury is an important cause of non-approval in drug development and the withdrawal of already approved drugs from the market. Screening human hepatic cell lines for toxicity has been used extensively to predict drug-induced liver injury in preclinical drug development. Assessing hepatic-cell health with more diverse markers will increase the value of in vitro assays and help predict the mechanism of toxicity. We describe three live cell-based assays using HepG2 cells to measure cell health parameters indicative of hepatotoxicity. The first assay measures cellular ATP levels using luciferase. The second and third assays are multiparametric high-content screens covering a panel of cell health markers including cell count, mitochondrial membrane potential and structure, nuclear morphology, vacuolar density, and reactive oxygen species and glutathione levels. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Measurement of cellular ATP content
Basic Protocol 2: High-content analysis assay to assess cell count, mitochondrial membrane potential and structure, and reactive oxygen species
Basic Protocol 3: High-content analysis assay to assess nuclear morphology, vacuoles, and glutathione content
Support Protocol 1: Subculturing and maintaining HepG2 cells
Support Protocol 2: Plating HepG2 cell line
Support Protocol 3: Transferring compounds by pin tool
Support Protocol 4: Generating dose-response curves
Cytidine acetyltransferases are an emerging class of nucleic-acid-modifying enzymes responsible for the establishment of N 4-acetylcytidine (ac4C) in RNA. In contrast to histone acetyltransferases, whose activity is commonly studied by western blotting, relatively few methods exist for quickly assessing the activity of cytidine acetyltransferases from a biological sample of interest or the distribution of ac4C across different RNA species. In this protocol, we describe a method for analysis of cellular cytidine acetyltransferase activity using dot- and immuno-northern-blotting-based detection. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Detection of N 4-Acetylcytidine in RNA by dot blotting
Basic Protocol 2: Visualizing N 4-Acetylcytidine Distribution in RNA by northern blotting
Small molecule microarray (SMM) technology has become a powerful tool used in high-throughput screening for target-based drug discovery. One area in which SMMs have found use is the identification of small molecule ligands for RNA. RNAs with unique secondary or tertiary three-dimensional structures are considered to be attractive targets for small molecules. Complex RNA structures can form hydrophobic pockets suitable for small molecule binding, representing an opportunity for developing novel therapeutics. Our lab has previously taken a target-based approach, screening a single target against many small molecules on an SMM platform. Here, we report a screening protocol for SMMs to investigate multiple RNAs simultaneously using multi-color imaging. By introducing a mixture containing different fluorophore-labeled RNAs, the fluorescence signal of each binding event can be observed simultaneously. Thus, the specificity of a hit compound binding to one RNA target over other highly abundant RNAs (such as tRNA or rRNA) can be easily evaluated. © 2020 Wiley Periodicals LLC.
Basic Protocol: RNA screening on SMMs by multi-color imaging
Support Protocol 1: Preparation of SMM slides
Support Protocol 2: Fluorophore labeling of RNA through maleimide chemistry
Reversible addition-fragmentation chain-transfer (RAFT) polymerization is a commonly used polymerization methodology to generate synthetic polymers. The products of RAFT polymerization, i.e., RAFT polymers, have been widely employed in several biologically relevant areas, including drug delivery, biomedical imaging, and tissue engineering. In this article, we summarize a synthetic methodology to display an azide group at the chain end of a RAFT polymer, thus presenting a reactive site on the polymer terminus. This platform enables a click reaction between azide-terminated polymers and alkyne-containing molecules, providing a broadly applicable scaffold for chemical and bioconjugation reactions on RAFT polymers. We also highlight applications of these azide-terminated RAFT polymers in fluorophore labeling and for promoting organelle targeting capability. © 2020 Wiley Periodicals LLC.
Basic Protocol 1: Synthesis of the azide derivatives of chain transfer agent and radical initiator
Basic Protocol 2: Installation of an azide group on the α-end of RAFT polymers
Alternate Protocol: Installation of an azide group on the ω-end of RAFT polymers
Basic Protocol 3: Click reaction between azide-terminated RAFT polymers and alkyne derivatives
Reverse-polarity activity-based protein profiling (RP-ABPP) is a chemical proteomics approach that uses nucleophilic probes amenable to “click” chemistry deployed into living cells in culture to capture, immunoprecipitate, and identify protein-bound electrophiles. RP-ABPP is used to characterize the structure and function of reactive electrophilic post-translational modifications (PTMs) and the proteins harboring them, which may uncover unknown or novel functions. RP-ABPP has demonstrated utility as a versatile method to monitor the metabolic regulation of electrophilic cofactors, using a pyruvoyl cofactor in S-adenosyl-L-methionine decarboxylase (AMD1), and to discover novel types of electrophilic modifications on proteins in human cells, such as the glyoxylyl modification on secernin-3 (SCRN3). These cofactors cannot be predicted by sequence, and therefore this area is relatively undeveloped. RP-ABPP is the only global, unbiased approach to discover such electrophiles. Here, we describe the utility of these experiments and provide a detailed protocol for de novo discovery, quantitation, and global profiling of electrophilic functionality of proteins. © 2020 The Authors.
Basic Protocol 1: Identification and quantification of probe-reactive proteins
Basic Protocol 2: Characterization of the site of probe labeling
Basic Protocol 3: Determination and quantitation of electrophile structure
Nature Methods (ISSN: 1548-7091) is a monthly journal publishing novel methods and significant improvements to basic life sciences research techniques. All editorial decisions are made by a team of full-time professional editors.
Nature Methods, Published online: 19 May 2025; doi:10.1038/s41592-025-02695-2
Bredt’s rule-inspired, bridged bicycle-strengthened fluorescent dyes resist both photobleaching and photoblueing, offering a powerful new toolkit for super-resolution fluorescence microscopy. This innovative design overcomes longstanding limitations associated with classical fluorescent dyes, enabling exceptional brightness and photostability — critical for capturing comprehensive spatial and temporal information over extended imaging sessions.Nature Methods, Published online: 19 May 2025; doi:10.1038/s41592-025-02693-4
A suite of bridged rhodamine dyes (BriDyes) offers excellent brightness, solubility, photostability, and tunable cell permeability along with resistance to photoblueing, making them exceptional all-purpose dyes for fluorescence biomaging.Nature Methods, Published online: 19 May 2025; doi:10.1038/s41592-025-02692-5
ALI is an approach for analyzing voltage imaging data that is inspired by algorithms used in super-resolution microscopy. It allows resolving the activity of single neurons in densely labeled populations in scattering conditions.Nature Methods, Published online: 15 May 2025; doi:10.1038/s41592-025-02701-7
Vclust generates fast and accurate estimation of average nucleotide identity for viral genomes, scaling clustering to millions of genomes.Nature Methods, Published online: 14 May 2025; doi:10.1038/s41592-025-02685-4
A universal AI-powered pipeline for fast, precise and plug-and-play image analysis of diverse 3D cellular structures across multiple imaging resolutions.Nature Methods, Published online: 13 May 2025; doi:10.1038/s41592-025-02715-1
Unraveling the black box in neural signal analysisNature Methods, Published online: 13 May 2025; doi:10.1038/s41592-025-02710-6
Tracking gene transfer using RNA toolsNature Methods, Published online: 13 May 2025; doi:10.1038/s41592-025-02709-z
Cell atlases for reproductive health
Nature Protocols (ISSN: 1750-2799 ) is an online journal of high-quality peer-reviewed protocols for researchers. Protocols are commissioned by the editors or submitted by authors as Presubmission Enquiries. They are presented in a 'recipe' style, providing step-by-step descriptions of procedures that users can take to the lab and immediately apply in their own research. All protocols have been proven to work already, having been used to generate data reported in published research papers.
Nature Protocols, Published online: 21 May 2025; doi:10.1038/s41596-025-01167-3
The Chemical Checker is a collection of small-molecule bioactivity signatures organized in increasing levels of biological complexity. This protocol describes how to incorporate new data, create novel bioactivity spaces and interpret the results.Nature Protocols, Published online: 19 May 2025; doi:10.1038/s41596-025-01165-5
This protocol enables rigorous and reproducible quantification of prokaryotic concentration in stool samples by 16S rRNA qPCR or ddPCR.Nature Protocols, Published online: 19 May 2025; doi:10.1038/s41596-025-01184-2
A closed-loop injectable bioelectronic patch, coupled to a robotic exoskeleton, forms a tissue prosthesis that maintains motor function in damaged muscle and enables early rehabilitation in rats.Nature Protocols, Published online: 14 May 2025; doi:10.1038/s41596-025-01144-w
In this Tutorial, we provide guidelines for the use of ML models to mine genomes and proteomes for the discovery of antimicrobial peptides, discussing the various stages of the ML workflow, recent advances in ML models and how to experimentally validate the computational predictions.Nature Protocols, Published online: 13 May 2025; doi:10.1038/s41596-025-01187-z
Nonsurgical, optically transparent windows noninvasively enable the visualization of cerebral hemodynamics, gut structure and motility and sarcomere structures in mice in vivo.Nature Protocols, Published online: 12 May 2025; doi:10.1038/s41596-025-01179-z
This Q-PHAST (Quantitative PHenotyping and Antimicrobial Susceptibility Testing) protocol provides a unique solution for cost-effective, large-scale phenotyping in a standard microbiology laboratory using affordable materials and open-source software.Nature Protocols, Published online: 29 April 2025; doi:10.1038/s41596-025-01168-2
This is a Protocol extension describing the establishment of human expanded potential stem cell lines from preimplantation embryos or by reprogramming somatic cells and their validation and characterization.Nature Protocols, Published online: 25 April 2025; doi:10.1038/s41596-025-01162-8
Studying the role of protein ubiquitination requires well-characterized ubiquitinated protein derivatives. This protocol describes a semisynthetic approach that can be used to make probes for reader proteins, deubiquitinases and ubiquitin carriers.For almost 30 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing.
Methods in Enzymology (ISSN: 0076-6879) a series of scientific publications focused primarily on research methods in biochemistry by Academic Press, created by Sidney P. Colowick and Nathan O. Kaplan, now part of Elsevier. Historically, each volume has centered on a specific topic of biochemistry, such as DNA repair, yeast genetics, or the biology of nitric oxide
Publication date: Available online 16 May 2025
Source: Methods in Enzymology
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Publication date: Available online 22 April 2025
Source: Methods in Enzymology
Author(s): Rosalie Lipsh-Sokolik, Paul J.N. Böhm, Clara Chepkirui, Jörn Piel
Publication date: Available online 16 April 2025
Source: Methods in Enzymology
Author(s): Hegne Pupart, Priit Eek
Publication date: Available online 15 April 2025
Source: Methods in Enzymology
Author(s): Vânia Brissos, Paulo Durão, Carolina F. Rodrigues, Eduardo P. Melo, Lígia O. Martins
Publication date: Available online 15 April 2025
Source: Methods in Enzymology
Author(s): José D.D. Cediel-Becerra, Marc G. Chevrette
Publication date: Available online 11 April 2025
Source: Methods in Enzymology
Author(s): Ana Serrano, Juan Carro
Publication date: Available online 2 April 2025
Source: Methods in Enzymology
Author(s): Gabriela Coy, Elliot N. Brajkovich, Elizabeth I. Parkinson
Publication date: Available online 19 March 2025
Source: Methods in Enzymology
Author(s): Awatif Alruwaili, Timothy D.H. Bugg
Publication date: Available online 18 March 2025
Source: Methods in Enzymology
Author(s): Lucas F. Ribeiro, Gilvan P. Furtado, Marcos R. Lourenzoni, Richard J. Ward
Publication date: Available online 12 March 2025
Source: Methods in Enzymology
Author(s): Wenbo Ning, Jeffrey D. Rudolf
Journal of visualized experiments [electronic resource] : JoVE
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