TABLE 3.
Purification of peak A and peak B signal molecules
| Step | Column | Flow rate (ml/min) | Elution method | Comment |
|---|---|---|---|---|
| 1 | 150-ml C18 Waters 125 A bulk packing materiala | 8 | 200 ml of 80% acetonitrile | |
| 2 | 50-ml C18 Waters 125 A bulk packing materiala | 1 | 0 to 80% acetonitrile gradient; 0.8%/ml over 100 ml | Two activity peaks |
| 3A | 4.6- by 250-mm Vydac 218TP C18 (peak A)a | 0.5 | 20 to 34% acetonitrile gradient; 0.18%/min over 75 min | Eluted at 28% acetonitrile |
| 3B | 4.6- by 250-mm Vydac 218TP C18 (peak B)a | 0.5 | 35 to 48% acetonitrile gradient; 0.18%/min over 75 min | Eluted at 42% acetonitrile |
| 4 | 1- by 150-mm Zorbax C18 (peak B only)b | 0.05 | 0 to 100% acetonitrile gradient; 0.25%/min over 400 min | Eluted at 40% acetonitrile |
a
Run and eluted in the presence of 0.02 M phosphate buffer at pH 7.
b
Run and eluted in the presence of 0.1% trifluoroacetic acid.