For experiments involving thyroid hormone (T3) induction of the f:TR-TRAP complex, stable f:TR-expressing HeLa cell lines (see Subheading 3.1.2.) should be expanded in Jokliks medium depleted of T3 for 3-6 d prior to hormone induction. Toward this end, we routinely expand the cells in dialyzed fetal serum (10,000 MW cut-off) (see Subheading 2.1.). Alternatively, one can deplete T3 from FBS manually by treating the serum with activated charcoal (Sigma) and the anion exchange resin AG1-X8 (Bio-Rad, Hercules, CA) (5).
1. Expand the f:TR-expressing HeLa-derived subline (Subheading 3.1.2.) into two 3000-mL spinner flasks, each containing 1.5 L Jokliks medium, 10% dialyzed FBS, 50 IU/mL penicillin and 50 |g/mL streptomycin per flask. When each culture reaches a density of 5 x 105 cells/mL, add 1.5 L of freshly prepared medium containing 2 x 10-7 M T3 to each flask (giving a final T3 concentration of 10-7 M) (see Note 11). Continue to grow the T3-induced cells for 48-60 h or until cell density reaches 5 x 105 cells/mL.
2. Harvest cells and prepare nuclear extract essentially as described in Subheading 3.2. Six L of cells (at 5 x 105 cells/mL) should yield about 5 mL of nuclear extract.
3. Thaw the nuclear extract (if frozen) on ice in a 4°C cold room. Adjust the KCl concentration in the nuclear extract to 300 mM using an appropriate amount of ice-cold 2.5 M KCl. Verify the KCl concentration in the extract using a conductivity meter; the conductivity should be roughly equivalent to that of the BC300 buffer.
4. In a 4°C cold room, mix 250 ||L (packed) pre-equilibrated M2 Affinity resin (see Note 12) with approx 5 mL of nuclear extract in a prechilled 5-mL culture tube (i.e., use 50 |L packed M2 resin per 1 mL nuclear extract). Securely seal the tube with parafilm and mix for 6-12 h (i.e., overnight) at 4°C on a rotator (one that rotates 360°).
5. Gently pellet the M2 resin in the cold room at 175g (1500 rpm) for 5 min using a swinging bucket rotor in a Centra CL2 clinical centrifuge (International Equipment Company, Boston, MA). Carefully remove the supernatant until approx 0.5-1 mL of supernatant remains above the pelleted M2 resin. Using a prechilled sterile transfer pipet, resuspend the M2 resin in the remaining nuclear extract and transfer the slurry into a sterile prechilled compact reaction column (CRC) containing a filter-plug but lacking a stopper. With the CRC tube attached to an upright support stand, let the nuclear extract drain out of tube by gravity allowing the M2 resin to pack inside the CRC tube (see Note 13). Insert a plastic stopper into the bottom of the CRC tube. The M2 resin should appear wet or moist. Do not let the resin dry out.
6. Attach a 5-mL syringe (minus plunger) to the top of the CRC tube using a leur-lock lid (supplied with the CRC tubes). Remove the plastic stopper and wash the packed M2 resin column with 5 mL prechilled BC300 buffer containing 0.1% NP40. Allow the buffer to drain out of the CRC tube by gravity and then repeat with a fresh 5 mL of BC300/0.1% NP40 (e.g., the M2 resin should be washed with approx 40 packed column volume of BC300/0.1% NP40). Finally, wash the column with 5 mL (approx 20 packed column volume) of freshly prepared BC100 buffer. Allow the buffer to completely drain out of the CRC tube and then insert a plastic stopper into the bottom of the tube.
7. To elute the f:TR-TRAP complex, add 250 |L elution buffer (BC100 buffer containing 0.2 mg/mL FLAG peptide) to the packed M2 resin (see Note 14). Securely attach a screw cap to the top of the CRC tube and mix the resin on a 360° rotator in the cold room for 1 h. Remove the plastic stopper from the bottom of CRC tube and (with cap partly unscrewed) place the CRC tube inside a prechilled microfuge tube. Collect the eluate by spinning at 175g (1500 rpm) for 10 s. Repeat the elu-
tion 1 or 2 more times (as above) by adding fresh BC100/FLAG peptide buffer to the M2 resin. Aliquot the eluted proteins in prechilled 0.5-mL microfuge tubes (approx 20 |L per tube) and then flash-freeze in liquid nitrogen. Store the extract at -80°C. The protein concentration of the TRAP complex (i.e., f:TR plus the TRAP subunits) is typically 20-40 ng/|L (with f:TR comprising about 10% of total protein). The f:TR-TRAP complex can be visualized by fractionating the proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (Fig. 3A) (see Note 15).
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