Long-Term Single-Molecule Tracking in Living Cells using Weak-Affinity Protein Labeling

ABSTRACT: The diffusion properties of the membrane proteins CD86 and CTLA4 were measured at 25 °C using single-particle tracking (SPT) in U 2-OS cells stably expressing CD86-HT7, CD86-dHT7, CTLA4-HT7, or CTLA4-dHT7. For imaging of the proteins of interest for 20 s, the covalent HaloTag Ligand SiR-HTL, or the exchangeable HaloTag Ligands (xHTLs) SiR-S5, SiR-Hy5, JF585-S5, JF585-Hy4 were used at a concentration of 1 nM. The proteins of interest were also measured for 20 min using SiR-HTL, SiR-S5, or SiR-Hy5 at the same concentration. The diffusion properties of the membrane receptor EGFR were measured at 25 °C using SPT in U-2 OS cells transiently expressing the EGFR-HT7 fusion protein. The ligand SiR-S5 was used for imaging the protein of interest for 20 s or 30 min at 1 nM concentration. The diffusion of calreticulin (CalR)-HT7-KDEL was measured in U-2 OS cells stably expressing the protein of interest and targeted with 0.5 nM SiR-HTL or SiR-S5 for 20 min.

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PROVIDER: S-BIAD1369 | bioimages |

REPOSITORIES: bioimages