Dynamic light scattering wyatt3/16/2023 We also found multiple factors, including pH, temperature, ionic strength, and protein concentration can affect LLPS behaviors. We found out that the droplets were formed through liquid-liquid phase separation (LLPS) as a result of protein self-association. These liquid droplets caused solution turbidity and exhibited extremely high viscosity, resulting in high column back pressure. A systematic study of three mAbs displaying this behavior revealed phase separation characterized by liquid drops under certain conditions including neutral pH, low ionic strength, and high protein concentration. We report several examples of turbidity and high column back pressure occurring transiently under a short course of neutral conditions during Protein A elution. In this work, we propose a new mechanism that may account for some observations of elution turbidity in Protein A chromatography. This phenomenon has been historically attributed to acid-induced precipitation of incorrectly folded or pH-sensitive mAbs and host cell proteins (HCPs). Turbid elution pools and high column back pressure are common during elution of monoclonal antibodies (mAbs) by acidic pH in Protein A chromatography. Our results indicate that in vivo NanoBRET is a tool to investigate specific protein interactions and localization in a physiological setting in real time in the living organism C. Using this approach, we traced ligand-induced internalization of NPR-11 in vivo. Furthermore, we adapted the enhanced bystander BRET technology to measure subcellular protein localization. We generated worms expressing NanoBRET sensors and elucidated the interaction of two ligand-G protein-coupled receptor (GPCR) pairs, the neuropeptide receptor NPR-11 and the Adhesion GPCR LAT-1. Since in vitro approaches mostly neglect the more complex in vivo situation, we established BRET as an in vivo tool for studying protein interactions in the nematode C. Bioluminescence resonance energy transfer (BRET) is a powerful tool to investigate these aspects in vitro. Protein-protein interactions form the basis of every organism and thus, investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligand-binding are of general interest.
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