Tandem Affinity Purification (TAP) Protocol (Page 3)
NP-40 Buffer (150 mL) add protease inhibitors and DTT
| Final Concentration | Volume | Stock |
|---|---|---|
| 15 mM Na2HPO4 | 4.5 mL | 500 mM |
| 10 mM NaH2PO4-H2O | 3 mL | 500 mM |
| 1.0% NP-40 | 1.5 mL | 100% |
| 150 mM NaCl | 4.5 mL | 5 M |
| 2 mM EDTA | 0.6 mL | 0.5 M |
| 50 mM NaF | 0.5 g | |
| 0.1 mM Na3VO4 | 150 µL | 100 mM |
Immediately before use, add inhibitors and adjust to pH 7.2 with NaOH.
| Protease Inhibitor | Stock | Volume |
|---|---|---|
| PMSF | 0.1 M in 100% isopropanol | 1 mL per 100 mL buffer |
| Benzamidine | 0.5 M in 100% water | 260 µL per 100 mL buffer |
| Pepstatin | 10 mg/mL in HAc | 100 µL per 100 mL buffer |
| Chymostatin | 5 mg/mL in HAc | 200 µL per 100 mL buffer |
| Aprotinin | 10 mg/mL in water | 100 µL per 100 mL buffer |
| Leupeptin | 10 mg/mL in water | 100 µL per 100 mL buffer |
Procedure is given on the next page.