Table 1.
Dynamics of eGFP and eGFP-LEDGF/p75 measured with sFRAP, FCS, CP, TFFCS and hnFRAP
| eGFP |
eGFP-LEDGF/p75 |
||||||
|---|---|---|---|---|---|---|---|
| sFRAP | FCS/CP | TFFCS | sFRAP | FCS/CP | hnFRAP | TFFCS | |
| Fimm (%) | 0.1 ± 0.1 | – | – | −1.2 | – | – | – |
| Dfast or DFRAP (µm2/s) | 10.28 ± 0.63a | 33.0 ± 3.5 | 36.4 ± 3.1 | – | 22.3 ± 4.1b | – | 35.3 ± 10.7 |
| Ffast (%) | – | 97.3 ± 0.7 | – | – | 35.9 ± 3.0c | – | 44.1 ± 5.7d |
| Dslow or DFRAP (µm2/s) | – | 0.8 ± 0.7 | – | 0.41 ± 0.01 | 0.5 ± 0.1 | 1.02 ± 0.01 | 0.95 ± 0.01 |
| αe | – | 0.98 ± 0.02 | – | – | f | – | |
| Ffree (%)g | – | – | – | 1.8 ± 3.1 | 2.2 ± 0.4 | 4.6 ± 0.94 | 4.3 ± 1.0 |
| Kdiss (nM) | – | 103 ± 66 | – | ||||
| [sites]high (nM) | – | 61 ± 31 | |||||
Values represent averages of at least 10 (sFRAP), 20 (FCS/CP), 10 (TFFCS) or 5 (hnFRAP) measurements and are ±SD.
aThis value is an underestimation, because the shortest bleach iteration (65 ms) was still too long to avoid recovery-while-photobleaching.
bThis parameter likely does not represent a diffusion process, see Figure 3F and ‘Discussion’ section.
cThe contribution of each component did not depend significantly on the concentration.
dThe fractional contribution of each component did not depend on the focus spot size.
eThis parameter was obtained by fitting with a one-component anomalous diffusion fit.
fFit not satisfying, see Supplementary Figure S2B.
gFfree is calculated from the apparent diffusion law, see ‘Materials and Methods’ section, Equation 7, with DeGFP-LEDGF/p75 = 22.3 µm2/s.
Fimm, immobile fraction; Dfast and Dslow, diffusion coefficient from fitting the FCS curves; DFRAP, diffusion coefficient obtained from Supplementary Equations S1 and S2; Ffast, fractional contribution of the fast component observed with FCS; α, anomaly parameter; Ffree, fraction of the protein population observed with FCS that is freely diffusing.