Magnetic Cytoskeleton Affinity (MICA) Purification for Fast Purification of Cargo-Attached Molecular Motors

Abstract

We have developed Magnetic Cytoskeleton Affinity (MiCA) purification method that is capable of isolating cargo attached molecular motors. We apply MiCA purification to the isolation of kinesin and compare with conventional microtubule affinity purification, which uses centrifugation to separate microtubules from the unbound or eluting kinesin. We show that MiCA purification generates purified kinesin yield comparable to that of the conventional microtubule affinity purification, and reduces the purification time from 1.5 hours to 0.5 hour, a 3 times improvement in speed. Using magnetic separation, we are now able to isolate quantum dot attached kinesin, which sediment together with microtubules upon centrifugation during conventional purification due to the dense quantum dot. This technique uses charge-charge interaction of the negatively charged microtubule and the positively charged magnetic beads to immobilize microtubule onto magnetic beads. Even though we have currently tested this MiCA purification technique only on cargo-attached kinesin, we foresee that the same protocol can be adjusted to purify cargo-attached dynein and other microtubule-associated proteins (MAPs). It also has the potential to purify cargo-attached myosin using the negatively charged actin instead of microtubule.