Volume 11 Issue 8, August 2016

Interaction with macromolecules affects the environmental availability of a micropollutant. With solid-phase microextraction of radiolabeled micropollutants, the partition coefficient can be determined at environmentally relevant concentrations.

SoNar is a fluorescent biosensor that is able to monitor NAD⁺/NADH redox state in living cells and in vivo. This protocol describes how to use SoNar for single cell imaging, high-throughput chemical screening, and in vivo imaging in mice.

This protocol describes the labeling and imaging of single adult neural stem cells and their progeny in zebrafish in vivo to investigate their contribution to neurogenesis in the intact and regenerating brain.

Cavitands are container molecules that have diverse applications from synthetic chemistry to molecular recognition. Deep, water-soluble cavitands based on a resorcinarene framework are prepared using this protocol.

Endo et al. describe a protocol for the isolation of mesophyll, vascular and epidermal tissues from Arabidopsis leaves. The protocol can be applied to tissue-specific transcriptome, methylome and proteome studies in a number of crop plants.

Nothobranchius furzeri is an important new model organism. This Protocol aims to help laboratories establish healthy breeding colonies of this species and to standardize husbandry methods, which differ from those for other model fish because of dry incubation of the eggs.

Labeling of proteins with fluorinated amino acids and using fluorine-19 NMR greatly simplifies the NMR spectrum. Changes in the spectrum on interaction with ligands makes protein-observed fragment-based drug screening possible.

Formalin-fixed paraffin embedding (FFPE) of tissues has been assumed to alter the metabolite content or chemical state, hampering metabolomics studies. Here, Ly et al. describe reproducible metabolomic analysis of FFPE samples by mass spectrometry imaging.

This protocol describes a fluorescence-based assay to measure cell death rate and type (either caspase-dependent apoptosis or caspase-independent necrosis). The method allows for the detection of modality switches between apoptosis and necrosis over time.

Mahat et al. describe how to map the genome-wide positions of active RNA polymerases using a modified nuclear run-on approach called PRO-seq. Details for PRO-cap, a modification that identifies transcription start sites, are also included.

This protocol enables simultaneous analysis of host and bacterial transcripts by RNA-seq. It includes procedures for efficient host and bacterial cell lysis, barcoding of samples and analysis of both mammalian and microbial reads from mixed host–pathogen RNA-seq data.

Cassaignau et al. provide a strategy for large-scale production and analysis of translationally arrested, isotopically labelled ribosome-bound nascent chains, enabling high-resolution co-translational protein folding studies using NMR spectroscopy.

Single-cell-resolution western blotting concatenates an electrophoretic separation to downstream immunoprobing of unfixed mammalian cells to detect proteins, isoforms, and interactions that are not discernable by immunoreactivity alone.

This protocol for bacterial flow cytometry incubates bacterial cells with an antibody-containing bodily fluid, visualizes bound antibody with secondary reagents and quantifies bacteria using flow cytometry, with numerous advantages over standard ELISA and western blotting techniques.

Magnani et al. describe a protocol to generate thermostable membrane proteins for structural analysis. This approach combines mutagenesis with a rapid, radioligand-based thermostability assay to screen and identify mutants with optimal stability.