September 2016-This Month in JoVE: Introducing JoVE Genetics, JoVE Biochemistry, and JoVE Cancer Research

Protocol

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Sarah A. Head¹, Jun O. Liu^1,2

¹Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, ²Department of Oncology, Johns Hopkins University School of Medicine

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

Hajime Miyaguchi

National Research Institute of Police Science

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/ mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Jason D. Domogauer, Sonia M. de Toledo, Edouard I. Azzam

Department of Radiology, New Jersey Medical School, Rutgers University

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

A Method of Targeted Cell Isolation via Glass Surface Functionalization

Ali Ansari¹, Reema Patel², Kinsey Schultheis¹, Vesna Naumovski³, P. I. Imoukhuede¹

¹Department of Bioengineering, University of Illinois at Urbana-Champaign, ²Department of Liberal Arts & Sciences, University of Illinois at Urbana-Champaign, ³Department of Biomedical Engineering, Illinois Institute of Technology

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Ai-Qun Yu^1,2, Nina Pratomo^1,2, Tee-Kheang Ng^1,2, Hua Ling^1,2, Han-Saem Cho^1,2, Susanna Su Jan Leong^1,2,3, Matthew Wook Chang^1,2

¹Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, ²NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, ³Food Science and Chemical Engineering, Singapore Institute of Technology

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

Yvonne Gunning¹, Andrew D. Watson¹, Neil M. Rigby², Mark Philo¹, Joshua K. Peazer^1,3, E. Kate Kemsley¹

¹Analytical Sciences Unit, Institute of Food Research, ²Institute of Food Research, ³School of Chemistry, University of East Anglia

We present a protocol for identifying and quantifying the components in mixtures of species possessing similar proteins. Mass spectrometry detects peptides for identification, and gives relative quantitation by ratios of peak areas. As a tool food for fraud detection, the method can detect 1% horse in beef.