Table 1.
Purification of Src-ΔU
| Step | Total activity, cpm ×10−9 | Total protein, mg | Specific actitivy, (cpm/mg) × 10−5 | Purification factor |
|---|---|---|---|---|
| Supernatant | 88 | 10,000 | 88 | 1 |
| Q-Sepharose 1 | 82 | 4,400 | 186 | 2.1 |
| Q-Sepharose 2 | 76 | 1,800 | 222 | 2.5 |
| ATP affinity | 66 | 35 | 18,860 | 214 |
| G75 gel filtration | 58 | 30 | 19,333 | 220 |
| MonoQ | 14 | 7 | 20,000 | 227 |
Kinase activity was measured in the presence of 1 mM activation peptide (phosphorylated middle T peptide) in 20 μl of reaction mixture and 100 μM of Src substrate peptide. The reaction was carried out for 3 min at 30°C; 15 μl of reaction mixture was pipetted on phosphocellulose units and washed twice with 75 mM phosphoric acid. The radioactivity bound to the filter was counted in a Beckman scintillator. Background incorporation obtained in the absence of enzyme was subtracted from all values.