Subcellular Localization of Intracellular Human Proteins by Construction of Tagged Fusion Proteins and Transient Expression in C
Identifying the subcellular compartment of a protein is an important step toward assigning protein function. Starting with a clone containing the open reading frame (ORF) of interest, it is possible to attach a variety of short amino acid tags or fluorescent proteins and detect the location of the protein, after transfection into a cell line, using fluorescent microscopy. By collecting data from various expression clone constructs, using a range of cell lines and double labeling with cellular compartment markers, a picture of the localization of a gene can be built up. This chapter describes how to obtain the ORF clone for your gene of interest, clone it into your choice of mammalian expression vector or vectors, transiently transfect for visualization, and where to get started when interpreting the results.
- Detection of RNA Editing Events in Human Cells Using High-Throughput Sequencing
- Targeted Gene Disruption with Engineered Zinc-Finger Nucleases (ZFNs)
- Plasmid Preparations with Diatomaceous Earth
- Purification of Supercoiled Plasmid DNA
- Mitochondrial Base Excision Repair Assays
- Physical Mapping of the Human Genome by Pulsed Field Gel Electrophoresis
- What Statisticians Should Know About Microarray Gene Expression Technology
- Flow Cytometry Analysis of an In Situ PCR for the Detection of Human Immunodeficiency Virus Type-1 (HIV-1) Proviral DNA
- Denaturing Gradient Gel Electrophoresis
- Capillary Electrophoresis of Multigene Barcoding Chloroplast Markers for Species Identification of Botanical Trace Evidence