Inverted Motility Assay: An In Vitro Technique to Visualize Myosin Movement on Immobilized Actin Filaments

Prepare the solutions for myosin 5a inverted motility assay, and keep them on ice.

Wash the chamber with 10 microliters of motility buffer with DTT. Flow in 10 microliters of 1 milligram per milliliter BSA in motility buffer with DTT. Repeat this twice, waiting a minute after the third wash. Use tissue or filter paper to wick the solution through the channel by gently placing the corner of the paper at the flow chamber exit.

Then, wash the chamber with 10 microliters of motility buffer with DTT three times. Flow in 10 microliters of the NeutrAvidin solution in motility buffer with DTT, and wait for one minute. Then, wash with 10 microliters of that solution three times.

Flow in 10 microliters of bRh-actin-containing motility buffer with DTT using a large-bored pipette tip, and wait for one minute. Then, wash with 10 microliters of motility buffer with DTT three times.

Flow in 30 microliters of final buffer with 10 nanomolar myosin 5a, and immediately load onto the total internal reflection fluorescence microscope. Begin recording once the optimum focus for TIRF imaging is found.