Effect of micro-viscosity on the rotational diffusion: pulsed laser based time resolved single molecule study

The effect of the changing in the buffer concentrations or any additives like surfactants to the protein samples during the analysis on the single bio molecule diffusion is one of the hidden points in the single molecule time resolved measurements. In the current work, PGK labeled with Atto 647 has been investigated on the single molecule level while it diffuses in Na 2 CO 3 buffer at concentrations that vary from 10 mg/l up to 50 mg/l. The fluorescence lifetime of PGK labeled with Atto 647 in 50 mg/ml Na 2 CO 3 has been measured, and it was found to be 2.7 ns. The Fluorescence cross correlation (FCS) of the diffused protein has also been measured and it confirms that the used samples are at a single molecule level. Time decay fluorescence anisotropy has been performed for PGK labeled with Atto 647 in different concentrations of Na 2 CO 3 , and the results confirmed that there is a clear impact on the molecular translational and rotational diffusion even with slight changes in the buffer concentration.