Table 2.

PRDM9 stoichiometry inferred based on FCS measurements.

ReplicatesNnativeNdenaturedNdenatured/NnativeAverage (CI)
Halo-eYFP-ZnFCst 1–1110.2580.9903.84
20.2420.8293.43
30.2950.9883.353.34 (0.41)
40.4191.2372.95
50.3561.1233.15
eYFP10.2280.3361.47
20.2350.2891.231.49 (0.66)
30.2290.4041.76
  • The free, soluble PRDM9 construct (Halo-eYFP-ZnFCst 1-11) was purified via ion-exchange chromatography and then measured by FCS in 1× TKZN buffer and 1× TKZN buffer + 3 M urea. Nnative and Ndenatured represents the brightness of single fluorescent particles in the focal volume without and with urea, respectively, estimated as described in Equation (1) in the Materials and Methods section.