Volume 9 Issue 7, July 2014

This Protocol describes how to downregulate specific plant genes using tobacco rattle virus virus-induced gene silencing (TRV-VIGS). The method can be used in a range of plants, but N. benthamiana is used here as an example.

The authors of this Protocol describe a system wherein human antigen-specific antibody secreting plasmablasts are enriched in vivo in a mouse host. The enriched plasmablasts can then be sorted by flow cytometry for subsequent IgG expression.

This article details a Protocol to achieve directed evolution of membrane proteins by displaying proteins on the surface of liposome membranes and iteratively selecting proteins with an enhanced activity of choice using a fluorescent indicator.

Clathrin is a protein complex that has roles in endocytosis and mitosis. This Protocol describes the synthesis of the Pitstop family of clathrin inhibitors and their inactive controls.

Creating transgenic flies is made quicker and easier with this Protocol from the Basler lab. Using ΦC31 integrase and pooled injections of 100 barcoded constructs, an entire library of transgenic flies can be generated in a few months.

A Protocol for obtaining high-quality protein crystals that involves addition to the protein solution of solid or semi-liquid nucleants that provide the experimenter the means to control crystal growth.

This Protocol covers the synthesis of NaGdF₄ nanoparticles that incorporate lanthanide ions in different layers and can be used as luminescent probes that efficiently convert near-infrared excitation wavelengths into tunable visible emissions.

Many physiological functions of helicases depend on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Here, Özeş et al. describe a fluorescence assay to monitor in real time RNA duplex unwinding by DEAD-box helicases.

A protocol on how to isolate c-kit-positive cardiac stem cells via magnetic activated cell sorting and how to then differentiate the cells into the three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle and endothelial cells.

CLARITY enables the chemical transformation of intact biological tissues into a hydrogel–tissue hybrid. The hybrid samples can be interrogated using light and macromolecular labels, whilst retaining fine structure and native biological molecules.

Anopholes gambiae has proven trickier to genetically manipulate than other mosquito species. Here, Pondeville et al. provide an optimized Protocol for ΦC31-mediated trangenesis that can generate transgenic A. gambiae in 2–3 months.

The authors of this protocol describe an approach that enables time-dependent analysis of dynamic processes in individual live cells for the quantitative investigation of signaling networks.

Biological questions that involve determining changes in protein structure that occur in solution may be answered using small-angle X-ray scattering. This protocol describes the sample preparation as well as the data collection and analysis.

TIF-Seq uses paired-end sequencing of genome-wide circular cDNA libraries to determine the 5′ and 3′ ends of each full-length transcript; the design maximizes intramolecular ligation to ensure that each read originates from a single transcript.

NGS error rates are very low in libraries prepared by CirSeq, allowing ultra-rare genetic variant detection in populations. Sequencing errors are easily identified as replicates of a particular sequence are physically linked during amplification.