Bioanalytical Research Group
iTAP Based Protein Complex Purification
Tandem Affinity Purification
The protocol was adapted from the original protocols developed in yeast1,2 and from Dignam et al.3.
1. Spin S2 cells at 200 g, 15 min., 4˚C
2. Wash cells with twice with PBS
3. Resuspend cell pellets with 5 x the pellet volume of Buffer A
4. Incubate on ice for 10- 15 min., to allow the cells to swell
5. Optional: Dounce with "B" type pestle at least 40x (s. Notes).
6. Spin at 700 g, 20 min., 4˚ C
7. Take off supernantant (don't discard!). This represents the cytoplasmic extract. Remove the nuclear contaminants by spining the cytoplasmic extract 10 min at 10 000g.
8. Resuspend the pellet from step 7 with 3-4x the pellet volume of Buffer B
9. Dounce with "B" type pestle at least 40x
10. Spin for 10 min at 10.000 g
11. Collect the supernatant. This represents the nuclear extract.
12. Nuclear and cytoplasmic extracts can be combined.
13. Equilibrate crosslinked IgG-beads (25-50 µl real beads) with 0.5 ml of Buffer B
14. Add extracts (nuclear or cytoplasmic or pooled extracts) to the IgG-beads and incubate for 2 hours at 4˚C using a rotating wheel.
15. Spin at 300 g, 1 min.
16. Take off supernatant
17. Wash three times with Buffer B
18. Wash four times with Buffer C
19. Wash twice with TEV Cleavage Buffer
20. Add 200 µl of TEV Cleavage Buffer and TEV protease (10 Units)
21. Incubate overnight at 4˚C using a rotating wheel.
22. Centrifuge: 300 g, 1 min.
23. Take off supernatant and add 0.6 µl 1 M CaCl2 per 200 µl supernatant
24. Add 3 volumes of Calmodulin Binding Buffer to the previous 200 µl TEV eluate
25. Equilibrate Calmodulin Resin (20-50 µl real beads) by washing twice with 1 ml Calmodulin Binding Buffer
26. Add TEV eluate to the Calmodulin Resin and incubate for at least 2 hours at 4˚C
27. Spin at 300 g, 1 min.
28. Take off supernatant.
29. Wash Resin five times with Calmodulin Binding Buffer
30. Elute by using SDS sample buffer (smallest possible volume)
- Depending on the fractionation of your protein you might only want to take the cytoplasmic or the nuclear fraction.
- If you plan to pool your fractions, you can either only lyse your cells in Buffer B or in addition dounce the cells in Buffer A to get a better solubilisation.
1. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17, 1030-2.
2. Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Seraphin, B. (2001). The tandem affinity purification (tap) method: a general procedure of protein complex purification. Methods 24, 218-29.