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Tandem
Affinity Purification (TAP) Protocol
Modified from Rigaut et al. 1999.*
Click here for PDF version, revised August, 2008.
Stock Solutions
| 100% NP-40 |
5 M NaCl |
1 M Mg Acetate |
| 100 mM DTT |
1 M Tris-HCl, pH 8.0 |
100 mM EGTA, pH 8.0 |
| 1 M Imidazole |
1 M CaCl2 |
500 mM Na2HPO4 |
| 14.3 M β-mercaptoethanol |
500 mM EDTA |
500 mM NaH2PO4 |
| BUFFERS |
| Note: For all buffers, add reducing agents
immediately before use! For some complexes it is beneficial to
add DTT to IPP300, IPP150 and NP-40 buffers. |
IPP300 (25 mL) add protease inhibitors (page 3) and DTT
|
Final
Concentration
|
Volume
|
Stock
|
| 25 mM Tris-HCl, pH 8.0 |
625 µL |
1 M |
| 300 mM NaCl |
1.5 mL |
5 M |
| 0.1% NP-40 |
0.25 mL |
10% |
| Milli-Q water to 100 mL |
IPP150 (10 mL) add protease inhibitors (page 3) and DTT
|
Final
Concentration
|
Volume
|
Stock
|
| 25 mM Tris-HCl, pH 8.0 |
0.25 mL |
1 M |
| 150 mM NaCl |
0.3 mL |
5 M |
| 0.1% NP-40 |
0.1 mL |
10% |
| Milli-Q water to 100 mL |
TEV Cleavage Buffer (TEV CB) (15 mL)
|
Final
Concentration
|
Volume
|
Stock
|
| 25 mM Tris-HCl pH 8.0 |
0.375 mL |
1 M |
| 150 mM NaCl |
0.45 mL |
5 M |
| 0.1% NP-40 |
0.15 mL |
10% |
| 0.5 mM EDTA |
15 µL |
500 mM |
| 1.0 mM DTT |
0.15 mL |
100 mM |
| Milli-Q water to 100 mL |
* Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Seraphin B. A generic
protein purification method for protein complex characterization and proteome
exploration. Nat Biotechnol 1999;17(10):1030-2.
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