Improve Research Reproducibility A Bio-protocol resource
- Protocols
- Articles and Issues
- For Authors
- About
- Become a Reviewer
Past Issue in 2025
Volume: 15, Issue: 15
Biochemistry
Direct Activity Measurement of Heterotrimeric Gi Proteins and Gq Protein By Effector Pulldown
Studying G protein-coupled receptor (GPCR) activation of heterotrimeric G proteins is crucial for understanding diverse physiological processes and developing novel therapeutics. Traditional methods to assay GPCR activation of G proteins, including assays of second messengers and biosensors, involve complex or indirect procedures. However, second messengers like cAMP and calcium are not direct readouts of GPCR activity due to signaling crosstalk, while biosensors can have undesired consequences due to structural alteration caused by fluorescent protein insertion. Here, we present a streamlined protocol employing GST-tagged bait proteins and epitope-embedded Gα subunits to achieve direct monitoring of Gα activity within cells. This method involves purification of GST-tagged bait constructs from bacteria and subsequent direct interaction studies with GluGlu-tagged Gα proteins expressed in any human cells of interest by including GST-tagged bait proteins in the cell lysis buffer. The approach enables sensitive detection of activated Gα within cells following extracellular stimulation. Advantages of this protocol include high sensitivity, enhanced monitoring of GPCR signaling dynamics under physiologically relevant conditions with minimum alteration in Gα, and the ability to distinguish between highly homologous isoforms within the same Gα family.
Reliable and Sensitive Detection of Carbonylated Proteins by Oxime Blot
Oxidative protein damage is important in various biological processes and age-related diseases. Protein carbonylation is the predominant and most frequently studied form of protein oxidation. It is most frequently detected following its derivatization with 2,4-dinitrophenylhydrazine (DNPH) hapten, followed by its detection with an anti-DNP antibody. However, when used to detect protein carbonylation by western blotting, this method suffers from diminished sensitivity, distortion of protein migration patterns, and unsatisfactory representation of low-abundance proteins. This is due to the poor solubility of DNPH in typical buffer solutions, the acidic protein precipitation due to the use of strong acid for its dissolution, the instability in solution, and the distorted protein migration patterns introduced by an additional salt content generated by the required pH adjustment prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To address the DNPH method limitations, a new Oxime blot technique was developed. This method is based on forming the stable oxime bonds between the protein carbonyl groups and biotin-aminooxy probe in the presence of a p-phenylenediamine (pPDA) catalyst at neutral pH conditions. The derivatization reaction reaches a plateau within 3 h. It ensures efficient and complete derivatization of carbonylated proteins, which are separated by SDS-PAGE without additional manipulation and detected with avidin-HRP and enhanced chemiluminescence (ECL) in western blotting. The Oxime blot protocol allows researchers to reliably and sensitively detect carbonylated proteins and provides a valuable tool for studying oxidative stress in diverse biological settings.
Bioinformatics and Computational Biology
Analyzing RNA Localization Using the RNA Proximity Labeling Method OINC-seq
Thousands of RNAs are localized to specific subcellular locations, and these localization patterns are often required for optimal cell function. However, the sequences within RNAs that direct their transport are unknown for almost all localized transcripts. Similarly, the RNA content of most subcellular locations remains unknown. To facilitate the study of subcellular transcriptomes, we developed the RNA proximity labeling method OINC-seq. OINC-seq utilizes photoactivatable, spatially restricted RNA oxidation to specifically label RNA in proximity to a subcellularly localized bait protein. After labeling, these oxidative RNA marks are then read out via high-throughput sequencing due to their ability to induce predictable misincorporation events by reverse transcriptase. These induced mutations are then quantitatively assessed for each gene using our software package PIGPEN. The observed mutation rate for a given RNA species is therefore related to its proximity to the localized bait protein. This protocol describes procedures for assaying RNA localization via OINC-seq experiments as well as computational procedures for analyzing the resulting data using PIGPEN.
Cell Biology
Reprogramming White Fat Cells for Adipose Manipulation Transplantation (AMT) Therapy
Adipocytes are endocrine cells that function as the main energy storage in our body. They are commonly used in clinical procedures, including their removal via liposuction and transplantation in plastic surgery. Building on this, adipocytes can be used for ex vivo cellular manipulations, enabling therapeutic modifications that can provide beneficial clinical outcomes after transplantation. Here, we provide a detailed protocol on how to modify adipocytes and adipose organoids using CRISPR activation (CRISPRa), a technology termed adipose manipulation transplantation (AMT).
Accurate Identification of Cell Cycle Stages in RPE1 Cells Using the ImmunoCellCycle-ID Method
Accurate identification of cell cycle stages is essential for investigating fundamental biological processes such as proliferation, differentiation, and tumorigenesis. While flow cytometry remains a widely used technique for such analyses, it is limited by its lack of single-cell resolution and its requirement for large sample sizes due to its population-based approach. These limitations underscore the need for alternative or complementary methods that offer single-cell precision with compatibility for small-scale applications. We present ImmunoCellCycle-ID, an immunofluorescence-based method that leverages the spatial distribution of endogenous markers, such as DNA, proliferating cell nuclear antigen (PCNA), centromere protein F (CENP-F), and centromere protein C (CENP-C), to reliably distinguish G1, early S, late S, early G2, late G2, and all mitotic sub-stages. This technique does not rely on precise signal quantification and utilizes standard immunofluorescence protocols alongside conventional laboratory microscopes, ensuring broad accessibility. Importantly, ImmunoCellCycle-ID detects endogenous proteins without the need for genetic modification, making it readily applicable to a wide range of human cell lines. Beyond its utility for single-cell resolution, the method can be scaled for population-level analyses, similar to flow cytometry. With its precision, versatility, and ease of implementation, ImmunoCellCycle-ID offers a powerful tool for high-resolution cell cycle profiling across diverse experimental platforms.
Developmental Biology
Fertility test of mice (Mus musculus)
Infertility has emerged as a global health concern, impacting around 8%–12% of couples during their reproductive years. Due to limitations in obtaining human biological samples, mouse models have been widely used for investigating gene functions. Fertility assessment in mouse models is a critical component in reproductive biology for studying gene function and elucidating mechanisms of reproductive disorders. However, natural mating observation of mice may yield inconsistent results, especially in the absence of standard guidelines, prolonged experimental cycles, and operational complexity. This protocol establishes a comprehensive breeding strategy for evaluating murine fertility through systematic vaginal plug monitoring and litter size quantification within defined timeframes. Key steps include (1) standardized male–female pairing protocols, (2) daily vaginal plug inspection, and (3) longitudinal tracking of pregnancy outcomes. This protocol presents a straightforward and easily implementable protocol for mouse mating cage setup and statistical analysis, enabling reliable fertility assessment under natural breeding conditions.
Neuroscience
Luminal Cerebrovascular Proteomics
Brain endothelial cells, which constitute the cerebrovasculature, form the first interface between the blood and brain and play essential roles in maintaining central nervous system (CNS) homeostasis. These cells exhibit strong apicobasal polarity, with distinct luminal and abluminal membrane compositions that crucially mediate compartmentalized functions of the vasculature. Existing transcriptomic and proteomic profiling techniques often lack the spatial resolution to discriminate between these membrane compartments, limiting insights into their distinct molecular compositions and functions. To overcome these limitations, we developed an in vivo proteomic strategy to selectively label and enrich luminal cerebrovascular proteins. In this approach, we perfuse a membrane-impermeable biotinylation reagent into the vasculature to covalently tag cell surface proteins exposed on the luminal side. This is followed by microvessel isolation and streptavidin-based enrichment of biotinylated proteins for downstream mass spectrometry analysis. Using this method, we robustly identified over 1,000 luminally localized proteins via standard liquid chromatography–tandem mass spectrometry (LC–MS/MS) techniques, achieving substantially improved enrichment of canonical luminal markers compared with conventional vascular proteomic approaches. Our method enables the generation of a high-confidence, compartment-resolved atlas of the luminal cerebrovascular proteome and offers a scalable platform for investigating endothelial surface biology in both healthy and disease contexts.
Isolation and Imaging of Microvessels From Brain Tissue
Proper brain function depends on the integrity of the blood–brain barrier (BBB), which is formed by a specialized network of microvessels in the brain. Reliable isolation of these microvessels is crucial for studying BBB composition and function in both health and disease. Here, we describe a protocol for the mechanical dissociation and density-based separation of microvessels from fresh or frozen human and murine brain tissue. The isolated microvessels retain their molecular integrity and are compatible with downstream applications, including fluorescence imaging and biochemical analyses. This method enables direct comparisons across species and disease states using the same workflow, facilitating translational research on BBB biology.
A Step-By-Step Protocol for Correlative Light and Electron Microscopy Imaging of Proteinaceous Deposits in Cultured Cells and Human Brain Tissues
An improved correlative light and electron microscopy (CLEM) method has recently been introduced and successfully employed to identify and analyze protein inclusions in cultured cells as well as pathological proteinaceous deposits in postmortem human brain tissues from individuals with diverse neurodegenerative diseases. This method significantly enhances antigen preservation and target registration by replacing conventional dehydration and embedding reagents. It achieves an optimal balance of sensitivity, accuracy, efficiency, and cost-effectiveness compared to other current CLEM approaches. However, due to space constraints, only a brief overview of this method was provided in the initial publication. To ensure reproducibility and facilitate widespread adoption, the author now presents a detailed, step-by-step protocol of this optimized CLEM technique. By enhancing usability and accessibility, this protocol aims to promote broader application of CLEM in neurodegenerative disease research.
Plant Science
Live Leaf-Section Imaging for Visualizing Intracellular Chloroplast Movement and Analyzing Cell–Cell Interactions
In response to environmental changes, chloroplasts, the cellular organelles responsible for photosynthesis, undergo intracellular repositioning, a phenomenon known as chloroplast movement. Observing chloroplast movement within leaf tissues remains technically challenging in leaves consisting of multiple cell layers, where light scattering and absorption hinder deep tissue visualization. This limitation has been particularly problematic when analyzing chloroplast movement in the mesophyll cells of C4 plants, which possess two distinct types of concentrically arranged photosynthetic cells. In response to stress stimuli, mesophyll chloroplasts aggregate toward the inner bundle sheath cells. However, conventional methods have not been able to observe these chloroplast dynamics over time in living cells, making it difficult to assess the influence of adjacent bundle sheath cells on this movement. Here, we present a protocol for live leaf section imaging that enables long-term and detailed observation of chloroplast movement in internal leaf tissues without chemical fixation. In this method, a leaf blade section prepared either using a vibratome or by hand was placed in a groove made of a silicone rubber sheet attached to a glass slide for microscopic observation. This technique allows for the quantitative tracking of chloroplast movement relative to the surrounding cells. In addition, by adjusting the sectioning angle and thickness of the unfixed leaf sections, it is possible to selectively inactivate specific cell types based on their size and shape differences. This protocol enables the investigation of the intercellular interactions involved in chloroplast dynamics in leaf tissues.
Systems Biology
Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution
Protein synthesis and degradation (i.e., turnover) forms an important part of protein homeostasis and has been implicated in many age-associated diseases. Different cellular locations, such as organelles and membraneless compartments, often contain individual protein quality control and degradation machineries. Conventional methods to assess protein turnover across subcellular compartments require targeted genetic manipulation or isolation of specific organelles. Here we describe a protocol for simultaneous proteome localization and turnover (SPLAT) analysis, which combines protein turnover measurements with unbiased subcellular spatial proteomics to measure compartment-specific protein turnover rates on a proteome-wide scale. This protocol utilizes dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) to resolve the temporal information of protein turnover and multi-step differential ultracentrifugation to assign proteins to multiple subcellular localizations. We further incorporate 2D liquid chromatography fractionation to greatly increase analytical depth while multiplexing with tandem mass tags (TMT) to reduce acquisition time 10-fold. This protocol resolves the spatial and temporal distributions of proteins and can also reveal temporally distinct spatial localizations within a protein pool.