Tandem Affinity Purification (TAP) Protocol (Page 1)

Modified from Rigaut et al. 1999.^*

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

Page 2

^*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.