Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published January 30, 2001 | public
Journal Article

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 μm diameter) over a density range comprising nearly four orders of magnitude (to 30 000 GFP/μm²). 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 >∼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%.

Additional Information

© 2001 Elsevier. Received 7 September 2000, Revised 30 October 2000, Accepted 30 October 2000, Available online 2 February 2001. We thank K. Beam for the GFP37 construct, N. Dinh and M. Young (City of Hope National Medical Center) for amino acid analysis, M. Simon for providing the Nikon microscope, and N. Davidson, R. Farley, K. Philipson, and B. Khakh for discussion. This work was supported by grants from the NIH (NS-11756, DA-09121).

Additional details

August 21, 2023
October 20, 2023