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  • Title: Methods for protein characterization by mass spectrometry, thermal shift (ThermoFluor) assay, and multiangle or static light scattering.
    Author: Nettleship JE, Brown J, Groves MR, Geerlof A.
    Journal: Methods Mol Biol; 2008; 426():299-318. PubMed ID: 18542872.
    Abstract:
    Mass spectrometry (MS) is widely used within structural and functional proteomics for a variety of tasks including protein quality assessment, identification, and characterization. MS is used routinely for the determination of the total mass of proteins, including N-glycosylated proteins, analysis of selenomethionine incorporation, crystal content verification, and analysis of N-glycosylation site occupancy. Protocols for sample preparation, data collection, and analysis are given.A recent development is the fluorescence-based thermal shift (ThermoFluor) assay. It uses an environmentally sensitive dye, Sypro Orange, to monitor the thermal stability of a protein and investigate factors (e.g., buffers, additives, and ligands) affecting this stability. This chapter describes the application of this method using a 96-condition in-house screen. The measurements are performed on a commercially available real-time PCR machine. Multiangle or static light scattering (SLS) is a very powerful technique to determine the conformational state of proteins in solution, especially when used in combination with size exclusion chromatography (SEC). In the authors' experimental set-up the SLS detector is connected in-line to a standard protein purification machine (e.g., the Akta Purifier) equipped with an analytical SEC column. The data collection and analysis are performed using commercial software.
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