LRRK2 RCKW Protein Purification

Ready the Ultracentrifuge and cool it down to 4°C

Make Lysis Buffer (check Materials section)

Thaw protein pellets On ice

Resuspend each pellet in 40mL

Homogenize each pellet with Dounce homogenizer On ice

15 plunges loose, followed by 17 plunges tight.

Pour lysate into rotor tubes (pre-chilled, clean, check for cracks; we used Ti70 tubes)

Make sure they are balanced. Use Lysis buffer to balance if needed.

Spin lysate in ultracentrifuge 50000rpm,4°C

Prep the rest of Day 1 buffers while waiting

Start equilibrating 12mL of Ni-NTA beads with Lysis buffer when there is about 30 mins left on the centrifuge

We equilibrated by resuspending 6mL in a 15 mL falcon tube, twice.

They were spun down at 1000rpm,4°C , each time getting rid of liquid and doing a 50:50 resuspension.

After ultracentrifuge is done, pour the supernate into 3x 50 mL falcon tubes.

Add 4mL to 40 mL of supernate. Bring up to 50mL

Incubate in cold room, while rotating "hot-dog style". rpm

After incubation, add onto a gravity column. Wash with remaining Lysis Buffer , followed by 200mL

Elute with 50mL

Make sure to resuspend the beads with stopper closed on the column. Might need multiple repeats of resuspension for best results. Leave column dry afterwards.

Dilute the solution to 250millimolar (mM) by adding 50mL

Syringe filter the solution.

Use a 5 mL HiTrap SP FF column. Pump wash FPLC with Buffer A and Buffer B . Equilibrate the column with Buffer A .

Apply sample onto the column with a sample pump

Run a gradient program, 250millimolar (mM) to 2.5Molarity (M) , 0% B to 100% B.

Combine protein fractions into a 50 mL Falcon Tube. Check protein concentration; should be around 1.1micromolar (µM)

Afterwards dilute to 600millimolar (mM)

Bring up to 50mL

Add TEV, and incubate overnight at 4°C while rotating "hot-dog style".

Make Day 2 buffers. Make sure to degas the Storage Buffer .

Put the 5 mL HisTrap column on the FPLC. Pump wash the FPLC with Buffer 0 and Buffer 300 . Equilibrate the column with Buffer 0 .

Run a Step Gradient Program

Combine protein elution into a 15 mL tube before concentrating for Size Exclusion Column

Equilibrate a S200I 10/300 column with the Storage Buffer .

Concentrate the protein to 1mL using 100 kD cutoff concentrators. Should be done nearing the end of the column equilibration.

After concentrating, filter the protein using a 0.1 uM spin filter. Spin at max speed for 0h 2m 0s at 4°C

Inject filtered sample and run elution program.

Collect and concentrate the protein to about 500µL . Concentration should be around 25-30micromolar (µM) after concentrating.

Aliquot and freeze protein. Make sure to check pureness of protein as well.