Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems.
Quantitative aspects of synaptic transmission can be studied by inserting green fluorescent protein (GFP) moieties into the genes encoding membrane proteins. To provide calibrations for measurements on synapses expressing such proteins, we developed methods to quantify histidine-tagged GFP molecules (His6-GFP) bound to Ni-NTA moieties on transparent beads (80-120 microm diameter) over a density range comprising nearly four orders of magnitude (to 30000 GFP/microm2). The procedures employ commonly available Hg lamps, fluorescent microscopes, and CCD cameras. Two independent routes are employed: (1) single-molecule fluorescence measurements are made at the lowest GFP densities, providing an absolute calibration for macroscopic signals at higher GFP densities; (2) known numbers of His6-GFP molecules are coupled quantitatively to the beads. Each of the two independent routes provides linear data over the measured density range, and the two independent methods agree with root mean square (rms) deviation of 11-21% over this range. These satisfactory results are obtained on two separate microscope systems. The data can be corrected for bleaching rates, which are linear with light intensity and become appreciable at intensities > approximately 1 W/cm2. If a suitable GFP-tagged protein can be chosen and incorporated into a 'knock-in' animal, the density of the protein can be measured with an absolute accuracy on the order of 20%.