To begin, prepare master buffer. Filter it using a 0.22 micrometer pore size filter, and store the buffer at 4 degrees Celsius. Next, prepare 75 milliliters of extraction buffer by supplementing the master buffer with three EDTA-free protease inhibitor tablets, 2 millimolar ATP, and 0.1 millimolar DTT.
Then add 75 milliliters of extraction buffer to the myosin VIIA-expressing frozen bacterial cell pellet, and leave the pellet in the extraction buffer on ice until it thaws. After that, use a serological pipette to break up the pellet to accelerate the thawing process. Now sonicate the resuspension on ice for 10 minutes at 50%amplitude using a half inch tip with 5 seconds on and 5 seconds off.
Centrifuge the total lysate at 33, 746 G for 30 minutes at 4 degrees Celsius. During the centrifugation, wash 3 milliliters of the 50%slurry of anti-FLAG affinity resin with 40 milliliters of PBS to prepare 1.5 milliliters of FLAG resin. Afterward, centrifuge the resin at 800 G for 2 minutes at 4 degrees Celsius.
Carefully remove the PBS without disturbing the resin. Then add the supernatant from the lysate centrifugation to the washed resin and incubate with continuous rotation at 4 degrees Celsius for 1 hour. Pellet the resin by centrifuging at 800 G for 2 minutes at 4 degrees Celsius.
And remove the supernatant without disturbing the resin. Next, re-suspend the resin in 40 milliliters of master buffer and centrifuge at 800 G for 2 minutes at 4 degrees Celsius. Carefully remove the supernatant without disturbing the resin.
Now transfer the washed resin to two polypropylene spin columns. Wash each column one time with 2 milliliters of master buffer. Then centrifuge the column at 1, 400 G for 2 minutes at 4 degrees Celsius to remove the master buffer.
To prepare an elution buffer, add 100 micrograms per milliliter of FLAG peptide to the master buffer. Add 300 microliters of elution buffer to the packed resin in each column. Gently mix by pipetting to ensure the resin is completely hydrated by the elution buffer.
Then centrifuge the columns at 1, 400 G for 2 minutes at 4 degrees Celsius. Transfer the flow through to a clean micro centrifuge tube and keep it on ice. Now, perform SDS-PAGE with the elution fractions to determine their relative concentrations.
While the gel runs, prepare a dialysis buffer with the given composition. Then combine the most concentrated fractions. Transfer the sample to a 10, 000 molecular weight cutoff dialysis cassette and dialyze overnight at 4 degrees Celsius.
The next day, carefully unload the sample from the dialysis cassette. Aliquot 15 microliters per PCR tube and drop the tubes into a container of liquid nitrogen for flash freezing. The purified myosin VIIA complex was confirmed by SDS-PAGE, showing a band above the 200 kilo Dalton marker, corresponding to the myosin VIIA heavy chain, and three distinct bands between the 22 and 14 kilo Dalton markers representing RLC calmodulin and calmodulin-like protein 4.
