Counting proteins bound to a single DNA molecule

Chromosomes contain DNA covered with proteins performing functions such as architectural organization and transcriptional regulation. The ability to count the number of proteins bound to various regions of the genome is essential for understanding both architectural and regulatory functions. We present a straightforward method of counting gfp-conjugated proteins bound to an individual duplex DNA molecule by calibrating to a commercially available fluorescence standard using wide-field fluorescence microscopy. We demonstrate our method using the E. coli nucleoid-associated protein Fis.

► Wide-field fluorescence calibration of fluorescence bead standard and bulk gfpFis. ► Number of proteins bound to a single DNA molecule is determined using calibration. ► Change in number of proteins bound as a function of force is measured.