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 g reen fluorescent protein (GFP) moieties into the genes encoding membrane pr oteins. To provide calibrations for measurements on synapses expressing suc h proteins, we developed methods to quantify histidine-tagged GFP molecules (His(6)-GFP) bound to Ni-NTA moieties on transparent beads (80-120 mum dia meter) over a density range comprising nearly four orders of magnitude (to 30 000 GFP/mum(2)). The procedures employ commonly available Hg lamps, fluo rescent microscopes, and CCD cameras. Two independent routes are employed: (1) single-molecule fluorescence measurements are made at the lowest GFP de nsities, providing an absolute calibration for macroscopic signals at highe r GFP densities; (2) known numbers of His(6)-GFP molecules an coupled quant itatively 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 sat isfactory results are obtained on two separate microscope systems. The data can be corrected for bleaching rates, which are linear with light intensit y and become appreciable at intensities > similar to 1 W/cm(2). If a suitab le GFP-tagged protein can be chosen and incorporated into a 'knock-in' anim al, the density of the protein can be measured with an absolute accuracy on the order of 20%. (C) 2001 Elsevier Science B.V. All rights reserved.