Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates
| Autor: | Aleksandra M. Urbanska, Pierluigi Reschiglian, Valerio Zolla, Cristina C. Clement, Laura Santambrogio, Jose A. Rodriguez-Navarro, Ana Maria Cuervo, Maya Tanase, Barbara Roda, Andrea Zattoni, Francesco Borghi |
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| Přispěvatelé: | Tanase, Maya, Zolla, Valerio, Clement, Cristina C., Borghi, Francesco, Urbanska, Aleksandra M., Rodriguez-Navarro, Jose Antonio, Roda, Barbara, Zattoni, Andrea, Reschiglian, Pierluigi, Cuervo, Ana Maria, Santambrogio, Laura |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Gel electrophoresis
Paraquat Biochemistry Genetics and Molecular Biology (all) Light Chemistry Medicine (all) Molecular biophysics Multiangle light scattering Hydrodynamic Fractionation Protein aggregation Cell Fractionation General Biochemistry Genetics and Molecular Biology Article Analytical Ultracentrifugation Protein Aggregates Biochemistry Biophysics Chromatography Gel Hydrodynamics Scattering Radiation Protein Aggregate Sample preparation Ultracentrifuge Ultracentrifugation |
| Popis: | Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. |
| Databáze: | OpenAIRE |
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