Prepare microcentrifuge tubes by adding mitochondria import retic lysate (I have been using up to 30ml) then add mitochondria and KRMs to desired concentrations, make sure to compensate with the appropriate buffer, mix well, THEN add the translation products.
24ml retic lysate for mitochondria import
56ml mitochondria @ 1-4 mg/mL
6ml FJ-41 KRM @ 4 eq/ml
2ml each translation product (Cb5, Bcl-2, pOCTgPA)
incubate 60 min. @ 30°c (mixing every once in a while)
place on ice for 5 min. to cool reactions.
make 0.5M in KAc and 50mM in EDTA (eg to the above reaction add about 12.3ml 4M KAc (pH 7.5) and 10.9ml 0.5M EDTA (pH 7.5)) mix well.
spin 5 min. 4°C, 10,000 rpm (Hermle, 1.5 ml tube rotor)
transfer supernatants to airfuge tubes containing 50ml of same sucrose cushion, spin 20 psi, 10min.
meanwhile remove remaining cushions from mitochondria tubes/ pellets and resuspend mitochondria pellets in 100ml resuspension buffer, spin 5 min. highest speed in mfuge at 4°C, remove supernatant and add 15ml 2xTricLoading buffer (2xTLB) to pellets, sonicate in bate sonicator for 30 sec, add 15ml 0.4M DTT, manually resuspend with pipet, heat at 80°C for 5 min.
from airfuge spin: remove and save supernatant, discard sucrose cushion, add 30ml TLB, heat 75°C for 10 min., resuspend manually
Retic Lysate for mitochondrial import
Translation products (Cb5, Bcl-2, pOCTgPA)
Mitochondria Buffer B (same as resuspension buffer)