Direct Quantification of PTD Transduction Using Real-Time Monitoring
Protein transduction domains (PTD or cell-permeable proteins) have attracted much attention as drug carriers because of their ability to penetrate cellular membranes. Although numerous PTD have been identified and their properties elucidated, their mechan
- PDF / 378,926 Bytes
- 8 Pages / 504.57 x 720 pts Page_size
- 33 Downloads / 146 Views
1
Introduction Protein transduction domains (PTD) have attracted increasing attention in intracellular therapeutic protein delivery (1), and quantifying cellular transduction of PTD is important for comprehending both their mode of transduction and the efficacy of new drugs (2). Current approaches for quantifying PTD transduction, including flow cytometry and confocal fluorescence microscopy, are based on the fluorescent labeling of peptides, but flow cytometry can lead to false-positive results originating from cell surfacebound peptides (3). Also, fluorescence imaging by confocal fluorescence microscopy, which can monitor the subcellular localization of peptides and discriminate between internalized and extracellular fluorescent peptides, is limited by statistical problems (4). Alternatively, direct peptide detection by MALDI-TOF using isotope-labeled peptide was recently reported (5, 6). Although this method allows for the direct quantification of cell-permeable
Volkmar Weissig et al. (eds.), Cellular and Subcellular Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 991, DOI 10.1007/978-1-62703-336-7_28, © Springer Science+Business Media New York 2013
307
308
Mi-Sook Lee and Song Her
Fig. 1 Schematic illustration of bioluminescence in living cells following PTD-Fluc transduction. Inset : Structures of PTD-Fluc containing 11 amino acids (YARVRRRGPRR) and Fluc as a control. After PTD-Fluc transduction, luminescence is emitted by an ATP-dependent luciferin–luciferase reaction (C cytosol, N nucleus)
proteins and the discrimination of extracellular membrane-bound and intracellular peptides, it can only be used with cell lysates and is not suitable for in vivo measurements. Bioluminescence imaging (BLI) based on luciferase activity is a rapid and sensitive method for in vitro and in vivo studies of ongoing biological phenomena. The most commonly used luciferase for BLI is firefly luciferase (Fluc), which requires ATP-Mg2+ and oxygen in the presence of the substrate, D-luciferin, to produce bioluminescence (7). Using firefly luciferase-tagged PTD (PTD-Fluc), we quantified internalized PTD by the real-time monitoring of ATP-dependent luciferase activity in vitro and in vivo (Fig. 1). This chapter provides a basic protocol for the real-time quantification of PTD transduction using BLI.
2
Materials
2.1 Luciferase Assay of Purified Protein
1. Purified PTD-Fluc (8) (see Note 1). 2. Purified Fluc (8) (see Note 1). 3. Luciferin substrate solution: Combine 1 mM D-luciferin (Xenogen, Alameda, CA, USA) (see Subheading 2.2), 3 mM ATP (ATP disodium salt), and 15 mM MgSO4 in 30 mM HEPES (pH 7.8) using fresh, deionized ATP-free water. Store the solution at −20°C in polypropylene or glass tubes. 4. 96-well black microplate with a clear bottom.
Real-Time Quantification of PTD Transduction
2.2 PTD-Fluc Transduction In Vitro
309
1. HeLa cells or other biologically relevant cell lines of interest. 2. Dulbecco’s modified Eagle’s medium (DMEM) complete media: DMEM supplemented with 10% (v/v) fetal b
Data Loading...
