Table 2. KD,app measurements for FANA binding to HIV-1 RT under different conditions.
| Buffer conditions used to measure KD,app | 1Approximate ionic strength (mM) | 2KD,app (pM) |
|---|---|---|
| Gel shift assay: 50 mM Tris–HCl pH 8.0, 80 mM KCl, 1 mM DTT, 2 mM MgCl2, 0.1 mg/ml BSA | 99 | 8±1 |
| Filter binding assay: | ||
| 50 mM Tris–HCl pH 8.0, 80 mM KCl, 1 mM DTT, 2 mM MgCl2, 0.1 mg/ml BSA | 99 | 4±3 |
| 50 mM Tris–HCl pH 8.0, 50 mM KCl, 1 mM DTT, 10 mM MgCl2, 0.1 mg/ml BSA | 93 | 16±8 |
| 1xPBS + Mg2+: 1.7 mM KH2PO4, 5 mM Na2HPO4, 150 mM NaCl, 2 mM MgCl2 | 170 | 22±4 |
| 50 mM Tris–HCl pH 8.0, 150 mM KCl, 1 mM DTT, 2 mM MgCl2, 0.1 mg/ml BSA | 190 | 74±20 |
1—Ionic strength was calculated assuming 50% ionization for Tris buffer as the pH was very close to the buffer pKa. An online calculator was used for the determinations (http://www.lenntech.com/calculators/activity/activity-coefficient.htm).
2—KD,app measurements were performed using nitrocellulose filter binding or gel shift assay as described in Materials and Methods. Results are an average of three or more independent experiment ± standard deviations.