Anti c myc agarose conjugated beads

Hypotonic purification of mitochondria from ~5 x 10⁷ uninduced and tet-induced PF cmyc-Tb927.1.1730, cmyc-Tb927.10.1730, and cmyc-Tb927.10.7910 cells was carried out as described above. The lysis buffer was prepared either with 40 U of RNaseOUT (Invitrogen) or 200μg/ml of RNase A from bovine pancreas (Sigma). Anti c-myc agarose conjugated beads (Sigma) were washed five times with 1ml ice-cold PBS at 4°C and subsequently washed once with 1ml of ice-cold immunoprecipitation wash buffer (Tris-HCL, pH 8.0, 10mM, NaCl 100mM, NP-40 0.1%, 1X complete EDTA-free protease inhibitor (Roch) and 1% PBS). After the last wash, 50 μl of beads were re-suspended for each reaction in 1 ml of ice-cold wash buffer and incubated for 1 hour at 4°C on a tube rotator. After adding the mitochondrial lysate to the beads, mixture was rotated for 2 hours at 4°C followed by centrifugation at 500rpm for 1 min at 4°C. After removing the supernatant (unbound proteins), the beads were washed four times with 1ml of immunoprecipitation wash buffer and then resuspended in SDS-PAGE loading dye. Aliquots of lysate, bound and unbound fractions were loaded on 10% SDS-PAGE gel. Proteins were transferred onto nitrocellulose membrane and probed with polyclonal antibodies against MRB 8170 and TbRGG2 (a generous gift from Laurie Read, state University of New York at Buffalo, USA).

Anti-C-Myc-agarose conjugated beads (Sigma-Aldrich) were pre-incubated in NT2 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% NP-40) supplemented with 5% BSA for at least 1 h before use. After several washes, beads were resuspended in 850 μL of ice-cold NT2 buffer with 200 units of RNase inhibitor (RNase Out, Invitrogen, Waltham, MA, USA), 400 mM of Vanadyl ribonucleoside complexes (Sigma-Aldrich), 10 μL of 100 mM DTT and EDTA to 20mM.