FIG. 3.
Purification of a recombinant Fu/Cos2 complex. Lysates of Sf21 cells infected with Cos2 and Flag G13V were purified over an anti-flag MAb (M2) affinity column and washed extensively. Bound protein was eluted with excess Flag peptide. The various fractions were resolved by SDS-PAGE and then Coomassie blue stained (A) or immunoblotted with Fu or Cos2 antibodies (B). (A) Coomassie blue-stained SDS-PAGE showing purification of a Cos2 Fu complex. Starting fractions containing the infected Sf21 lysate (Lysate), the high speed supernatant (HSS), the flowthrough fraction from the Flag M2 affinity resin (Flow-through), and the fractions collected after addition of excess Flag peptide are shown (lanes 1 to 13, every other fraction). Two major proteins that correspond to the apparent molecular weights of Fu and Cos2 are seen in lanes 5 to 9. (B) Immunoblot analysis of the Cos2 Fu purification. Fu and Cos2 immunoreactivity comigrated with the two major Coomassie blue-stained proteins, at molecular weights of 83,000 and 175,000. (C) Fraction 8 off the M2 affinity resin was loaded onto a Superose 6 gel filtration column to determine quantitative association between purified Cos2 and G13V. Both purified proteins comigrated at fraction 32, verifying that Fu and Cos2 are sufficient to form the bulk of peak B seen in S2 lysates.