Table 2.
Improvements in Tm variability made through buffer optimization. Proteins are from P. falciparum unless otherwise noted. Results in the fourth column are based on comparisons of the standard buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) and optimal buffers side-by-side on the same plate (8–16 wells per buffer treatment per plate). "N" in the fourth column is the number of plates run for that protein.
| Protein | PlasmoDB ID | Optimal buffer | Improvement vs. standard buffer |
|---|---|---|---|
| 6-pyruvoyltetrahydropterin synthase |
PFF1360w | 100 mM citric acid, pH 5.5, 100 mM NaCl |
Decreased mean standard deviation in Tm from 2.7 to 0.4 °C (N=6). |
| Adenosine deaminase | PF10_0289 | 100 mM Bis-Tris, pH 6.0, 100 mM NaCl, 10% Glycerol |
Increased mean Δfluorescence from 0.05 to 1.73 (arbitrary units), permitting Tm's to be measured in all wells (N=3). Some wells were uninterpretable when using standard buffer. |
| Adenosine deaminase (P. vivax) |
PVX_111245 | 100 mM potassium phosphate, pH 7.0, 500 mM NaCl, 10% Glycerol |
Decreased mean Δtemperature25–75 from 8.9 to 3.9 °C and decreased mean standard deviation in Tm from 1.1 to 0.3 degrees (N=9). Increased mean Z'-factor from −0.10 to 0.47 (N=6). |
| Phosphoethanolamine N-methyltransferase |
MAL13P1.214 | 100 mM MOPS, pH 7.0, 100 mM NaCl |
Increased mean Δfluorescence from 0.07 to 0.23 (arbitrary units), permitting Tm's to be measured in all wells (N=3). Some wells were uninterpretable when using standard buffer.. |
| Superoxide dismutase (P. berghei) |
PB000490.02.0 | Standard buffer + 1 mM MnCl2 |
Decreased mean Δtemperature25–75 from 8.8 to 4.6 °C and decreased mean standard deviation in Tm from 1.0 to 0.3 °C (N=10). Increased mean Z'-factor from 0.39 to 0.75 (N=6). |
| Superoxide dismutase (P. knowlesi) |
PKH_142350 | Standard buffer + 1 mM MnCl2 |
Decreased mean Δtemperature25–75 from 11.4 to 5.5 °C and decreased mean standard deviation in Tm from 3.1 to 0.4 °C (N=7). Increased mean Z'-factor from −0.61 to 0.68 (N=6). |