Bioanalytical Research
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.
Protocol:
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)
Notes:
- 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.