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PCR Cloning and Subcloning> | … PGK-Hsp70 binding in the cell. A fluorescent hsp70 fusion protein was created by cloning the sequence for the human, cytoplasmic, heat-inducible hsp70 (hsp72) [30] with a C-terminal mCherry tag into the pDream 2.1/MCS vector (Genscript Corp., Piscataway, NY) … | Get A Quote |
When a protein unfolds in the cell, its diffusion coefficient is affected by its increased hydrodynamic radius and by interactions of exposed hydrophobic residues with the cytoplasmic matrix, including chaperones. We characterize protein diffusion by photobleaching whole cells at a single point, and imaging the concentration change of fluorescent-labeled protein throughout the cell as a function of time. As a folded reference protein we use green fluorescent protein. The resulting region-dependent anomalous diffusion is well characterized by 2-D or 3-D diffusion equations coupled to a clustering algorithm that accounts for position-dependent diffusion. Then we study diffusion of a destabilized mutant of the enz... More