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

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The contraction cycle of muscles involves the binding of myosin to actin, which is regulated by the presence of ATP and calcium ions. Initially, myosin is unable to bind to actin without ATP. ATP binds to myosin, causing it to detach from actin if previously bound, and upon hydrolysis to ADP + Pi, myosin is energized and can bind to actin again. Therefore, in the context of purifying myosin using an actin column, the first wash should not include ATP to allow myosin to bind to the immobilized actin. The second wash should include ATP to release the myosin from the actin, allowing it to be collected. This makes use of the natural interaction between myosin and actin regulated by ATP, without the need for calcium, which primarily regulates the exposure of the binding site on actin in muscle cells.

One Way One Can Purify Myosin from Other Skeletal Muscle