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Synthetic ion channels are used to study biological processes and develop sensing, drug delivery and antimicrobial systems. This protocol uses click chemistry to prepare lipophilic diguanosine derivatives that assemble into metal cation channels.
Here the authors detail the protocol for their recently developed liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy to enable the systematic and quantitative measurement of protein–lipid interactions.
The use of a single enzyme such as trypsin for shotgun proteomics limits the ability to cover the whole proteome and all protein post-translational modifications. This protocol describes the use of six alternative proteases that complement trypsin to increase the coverage of the proteome.
This protocol for the induction and isolation of Plasmodium falciparum gametocytes combines seven parameters that have been shown to facilitate the optimum induction of gametocytogenesis in vitro to obtain highly synchronous gametocyte stages on a large scale.
Here the authors provide the procedural details of a new approach to clone large (up to 100 kb) microbial genomic sequences using Cas9-assisted targeting of chromosome segments (CATCH). The Cas9 cleavage is achieved in agarose gel, and the cleaved product can be ligated into a cloning vector.
This protocol describes a rapid (<24 h), reproducible and cheap technique for evaluating cancer cell invasiveness by quantifying in vivo rates of cancer cell extravasation in the chorioallantoic membrane (CAM) of chicken embryos.
This protocol provides a method for genetically engineering untransformable coagulase-negative staphylococci (CoNS), using bacteriophage Φ187–mediated plasmid transfer. This is a major technical advancement that enables research on CoNS-mediated infections.
This protocol describes the use of the AutoDock suite for computational docking in the study of protein–ligand interactions. A number of methods are described ranging from basic docking of drug molecules to virtual screening using a large ligand library of chemical compounds.
MicroED is a cryo-EM technique for collecting electron diffraction data from microcrystals and nanocrystals. This protocol from Gonen and colleagues describes how to prepare the protein crystal samples, how to set up the electron microscope for MicroED, and diffraction data collection.
This protocol describes how to establish explant cultures of intact zebrafish hearts. The explants can be used to study the regeneration of cardiac tissues such as the epicardium, can be maintained for up to 30 d and are amenable to live imaging.
This protocol from Brautigam et al. describes methods for baseline correction and global analysis of isothermal titration calorimetry data using NITPIC and SEDPHAT. Publication-quality graphs of resulting data can then be created and visualized using GUSSI.
This protocol describes a genome-wide method to detect and to quantify DNA double-stranded breaks (DSBs). The approach is applicable to endogenous DSBs, but it can also be used to characterize the activity of engineered nucleases, including Cas9.
Human NET-seq enables DNA strand–specific mapping of RNA polymerase (RNAP) activity at single-nucleotide resolution. A cell fractionation approach is used to isolate transcribing RNAP and associated RNAs, avoiding immunoprecipitation or RNA labeling.
Many solid tumors contain an aggressive hypoxic region that is difficult to treat. This protocol describes how to prepare bioreductive prodrugs that are biologically inactive until they are converted to an active drug by enzymatic reduction in hypoxia.
This protocol describes a data-independent acquisition workflow for label-free quantitative proteomics that integrates ion mobility separation and applies drift time–specific collision energies to improve precursor fragmentation efficiency.
This protocol describes how to make semisynthetic gelatin methacryloyl (GelMA)-based hydrogels for use in 3D cell culture models for cancer research, stem cell research and tissue engineering.
This protocol describes an approach for screening DNA-encoded chemical libraries (DECLs) to identify molecules that bind to proteins of interest. After isolating binding library members, DNA barcodes are amplified and identified by high-throughput sequencing.