One way one can purify myosin from other skeletal muscle proteins is to immobilize actin on a ...

One way one can purify myosin from other skeletal muscle proteins is to immobilize actin on a column, pour a mix of the muscle proteins through, wash first to get out the nonspecific proteins and then do a second to wash to obtain myosin. Based on the contraction cycle, your first wash should
 a. include ATP, but the second wash should not.
  b. not include ATP, but the second wash should.
  c. include ATP, as should the second wash.
  d. not include ATP, and neither should the second.
  e. should include calcium, but the second one should not.