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Phosphatidylethanol (PEth) is a metabolite of ethanol that can be used as a stable, direct marker of alcohol usage. This protocol describes how to determine the concentration of PEth in dry blood spots via manual or automated extraction followed by LC-MS/MS.
Delivery of siRNAs to immune cells is challenging because of these cells’ sensitivity to standard transfection reagents. This protocol describes how to synthesize an amphiphilic dendrimer and apply it to transfect siRNAs in a variety of primary immune cells.
This is a protocol for the extraction of microgram quantities of high-molecular-weight DNA from human stool samples that are suitable for downstream long-read sequencing applications.
This protocol describes the construction, calibration and use of the Drosophila traumatic brain injury (dTBI) device to induce different degrees of TBI in flies. Two versions of the device are described, for low- and high-throughput applications.
Mechanical sample motion (drift) is a major factor that limits fluorophore localization precision during single-molecule localization microscopy. This protocol describes how to implement 3D active stabilization onto custom and standard microscopes.
This protocol describes a strategy for revealing the initial interactions between host proteins and incoming viral RNAs that relies on solid-phase purification of crosslinked RNA–protein complexes followed by mass spectrometry.
Here, we present a protocol for producing exosomes loaded with ultrathin Pd nanosheets for targeted bio-orthogonal catalysis. Pd precursors are loaded into exosomes by diffusion and reduced into metallic nanosheets by using gas-phase CO.
Culture conditions are described for long-term expansion of human fetal hepatocytes as 3D organoids. Gene knockin and knockout approaches are also described for organoids derived from human fetal hepatocytes and human adult liver ductal cells.
This protocol describes how to perform antigen retrieval and tissue clearing for volumetric imaging of whole organs, organoids and small organisms by using fast light-microscopic analysis of antibody-stained whole organs.
This protocol describes how to convert a commercial confocal spinning-disk microscope into an image scanning microscope (ISM), allowing fast imaging of fixed and live samples with increases of up to twofold in resolution and fourfold in contrast.
This extension of the original silicon fluoride acceptor (SiFA) protocol for 18F-labeling of peptides describes modifications required for the preparation of clinical-grade [18F]SiFAlin-TATE for diagnosis of neuroendocrine tumors via PET imaging.
This protocol describes experimental and computational procedures for obtaining genome-wide DNA replication timing maps based on copy-number differences derived from whole-genome amplification and next-generation sequencing of genomic DNA from single S-phase cells.
This protocol extension describes an improved method for global profiling of poly(A) RNA-binding proteins (RBPs) and quantitative analysis of RBP dynamics in response to biological and pharmacological cues that uses UV crosslinking, capture with LNA-modified oligo-dT probes, and proteomics.
This phage-assisted continuous evolution protocol enables directed protein evolution. It includes preparation of selection phage and host cells, assembly of a continuous flow apparatus and performance and analysis of evolution experiments.
This protocol describes a comet-based in vitro assay for detecting base and nucleotide excision repair activity for use in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues.
Footprinting reports solvent accessibilities of a protein or protein complex. This protocol describes a fast photochemical oxidation of proteins platform for footprinting, with laser-initiated H2O2 photolysis and mass spectrometry analysis.
The authors describe procedures for generating and biobanking glioblastoma organoids from patient tumor tissue and testing of chimeric antigen receptor T cell efficacy by co-culture. Tissue processing, immunohistology and detection of hypoxic gradients and actively proliferating cells are also described.
This protocol describes proximity labeling approaches using TurboID and split-TurboID, which can be used for mapping protein–protein interactions and organelle proteomes in live mammalian cells with nanometer spatial resolution.
This protocol describes experimental and computational procedures for genome-wide detection of endogenous and induced DNA double-strand breaks (DSBs) in any cell type or tissue that can be brought into suspension.