Tandem Affinity Purification (TAP) Protocol (Page 4)

Procedure

  1. Grow 2 L of yeast to 150 Klett units (4.5×107 cell/mL).
  2. Pellet cells at 13,300 g for 5 min in Beckman Avanti J-25 centrifuge using the JA-9.1000 rotor and the 1 L centrifuge bottles.
  3. Wash 1× with NP-40 buffer while transferring to 40 mL centrifuge tubes.
  4. Pellet cells at 5000 g for 5 min in the JA 25.50 rotor.
  5. Resuspend the cells in about 10–15 mL of NP-40 buffer and transfer to the 50 mL chamber of the bead beater.* Add glass beads† to about half to two-thirds full. Fill almost to the brim with NP-40 buffer. Pop air bubbles using a pasteur pipette and bulb. Attach cap and limit the amount of air trapped inside. Attach the outer chamber, place in an ice bath, and lyse cell 1 min on/1 min off, 10×, with a 5-min "off" period half-way through. Refill outer chamber with ice every third round.
  6. Check lysis by microscope. If <90%, do a few additional rounds.
  7. Transfer lysate to 40 mL plastic centrifuge tube. Clarify at 5000 g for 10 min in a JA 25.50-rotor Beckman Avanti J-25 centrifuge.
  8. Transfer crude lysate to tubes fitting the Type 45-Ti ultracentrifuge rotor. On a scale, adjust volume such that it almost fills the tube. Make sure opposite tubes are well balanced.
  9. Spin lysates at 142,000 g for 1.4 hr at 4°C in a Beckman ultracentrifuge.
  10. Retrieve the supernatant to a 50 mL Falcon tube. (Avoid disturbing the pellet and avoid collecting the top lipid layer.)
  11. Add 1 mL of Sepharose 6B (Sigma) beads prepared in a 1:1 slurry with NP-40 buffer. Incubate on a rotating platform at 4°C for 30 minutes. Spin in a tabletop centrifuge to pellet the beads. Retrieve the supernatant into a new 50 mL Falcon tube.
  12. Adjust NaCl concentration to 300 mM with 5 M NaCl.
  13. Add 500 µL of IgG Sepharose 6 Fast Flow (Amersham)(previously prepared in a 1:1 slurry with NP-40 buffer) and incubate on a rotating platform at 4°C for at least 2 hr.
  14. Pour the lysate/IgG Sepharose suspension onto a chromatography column (BioRad Poly-Prep) with a reservoir. Pull through with the pump.
  15. Wash beads 2× with 10 mL of IPP300 buffer and 1× with 10 mL of IPP150 buffer.
  16. Wash beads with 10 mL of TEV CB.
  17. Close the bottom with a stopper and add 1 mL of TEV CB and 5 µg of TEV. Plug the top of the column and incubate on a rotating platform between 2 hr and overnight at 4°C.
  18. Drain eluate into a new column sealed at the bottom.
  19. Wash out old column with 1.0 mL of TEV CB.
  20. Add 3 vol (6 mL) of CBB to the TEV supernatant. Plus 3 µL of 1 M CaCl2 per mL of IgG eluate (~6 µL). Add 300 µL of calmodulin Sepharose 4B (Amersham) in CBB (1:1 slurry) and incubate on platform for 1 hr at 4°C.
  21. Wash:
    •  Wash 2× in 1 mL CBB (0.1% NP-40).
    •  Wash 1× in 1 mL CBB (0.02 % NP-40).
  22. Plug the bottom of the column and add 1 mL of CEB.
  23. Elute first 1 mL fraction into a silconized microfuge tube.
  24. Plug the bottom of the column and add 1 mL of CEB.
  25. Elute second 1 mL fraction into a silconized microfuge tube.
  26. Combine the fractions and split into 500 µL, 750 µL, and 750 µL into non-silconized microfuge tubes.
  27. Adjust aliquots to 25% TCA with 100% TCA and place on ice for 30 min with periodic vortexing.
  28. Spin at maximum speed (13,000 rpm) in a microfuge at 4°C for 30 min.
  29. Wash 1× with ice cold (-20°C) acetone containing 0.05 N HCl, and spin 5 min at maximum speed (13,000 rpm) at 4°C.
  30. Wash 1× with ice cold (-20°C) acetone, and spin 5 min at maximum speed at 4°C.
  31. Remove supernatant and dry in a speed vacuum for ~10 min.
Use the 500-µL fraction for silver stain on a 10% gel.
Send one 750-µL fraction for mass spectrometry.
Save one 750-µL fraction just in case.
* BioSpec Products, Inc., cat. no. 1107900.
† 425–600 µm. Wash with nitric acid before using; rinse until ph >6.0.

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