Tnt sp6 quick coupled transcription translation system

For pull down assays, recombinant His-Mta2 and His-Tipin were purified using Ni-NTA beads. Mta2 was also produced in vitro using the Sp6 TNT-quick coupled transcription/translation system (Promega). Binding reactions contained 2 μg his-tagged recombinant protein and 15 μl the in vitro translated protein in 1 ml binding buffer (20 mM TRIS-HCl pH 7.5 200 mM NaCl 0.5% NP40). The reactions were incubated for 3 h at 4 °C, followed by 5 washes in binding buffer. Complexes were resolved by SDS/PAGE and probed with the indicated antibodies or exposed to the phosphoimager for detection of the ³⁵S signal. For the in vitro pull down shown in Figure S2C 2 µg recombinant His-Tipin protein were incubated for 1 h at 4 °C in binding buffer with either recombinant His-MTA2 (500 ng) or His-Geminin (1 µg) used as negative control. The mixture was then supplemented with 1 µg mouse anti-Tipin antibody or mouse IgG as control. One hour later, 15 µl protein A/G agarose beads (Santa Cruz) were added to reaction and left incubating for 1 h at 4 °C. Next, beads were washed 4 times with coupling buffer and once more with PBS before eluting proteins in Laemmli buffer. Samples were then resolved on a SDS-PAGE and analyzed by western blotting with anti-MTA2 antibody.