Detection of accessible cholesterol in primary cilia using purified His-ALOD4-mNeon in 3T3 Fibroblasts

Make a 50mL starter culture of BL21 DE3 Rosetta plysS cells expressing pRSET+ His-mNeon-FLAG-ALOD4, starting from a fresh colony picked from freshly transformed plates containing carbenicillin (100μg/ml) and chloramphenicol (34μg/ml). Grow with shaking in an Erlenmeyer flask at 37°C.

Day 2. Using a 6 liter flask, prepare 2 X 2 liters LB by mixing 50g LB powder with 2L water. Autoclave for 0h 20m 0s using a liquid cycle. Allow the media to cool to Room temperature

Add carbenicillin(100µg)and chloramphenicol (34µg) to each flask and shake at 37°C until an OD 0.5-0.6 is reached. This usually takes ~2.5 hr.

Transfer flask to an 18°C shaker and equilibrate temperature for 10-20 min.

Add IPTG to achieve a concentration of 0.3millimolar (mM)

Induce expression overnight with shaking at 180rpm at 18°C

Day 3. Transfer bacterial culture to 4, 1L centrifuge bottles.

Pellet bacteria by spinning at 4000 RPM for 20 min 4°C; discard supernatant.

Add 1 Roche protease inhibitor tablet to 50mL of lysis buffer, rotate at 4°C to dissolve.

Set up a ~200mL beaker with a stir bar at 4°C in the cold room.

Resuspend pelleted bacteria On ice or in cold room using ~10mL of lysis buffer for each bottle.

Vortex the bottles to help resuspend pellet; transfer resuspended pellets to the beaker with a stir bar.

Take 40mL of this suspension to the Emulsiflex cell breakage device and have ready, 4 x 65ml polycarbonate ultracentrifuge tubes on ice for the homogenate from the next step. Pass 10mL lysis buffer through the Emulsiflex once at 0-30K PSI to equilibrate the machine. Pass suspension through the Emulsiflex once at ~50K PSI to lyse cells. Collect directly into polycarbonate tubes On ice.

Balance tubes and then spin at 40,000 RPM for 0h 45m 0s in a 45Ti rotor at 4°Cto pellet cell debris. Collect supernatant and filter through a 500mL, 0.45µm Millipore filter. Collect the filtered supernatant in a 100mL glass beaker On ice.

Take a 1mL HiTrap Talon column (Cytiva #28953766) and wash it with 10 column volumes (CV) of distilled, degassed water. Equilibrate column with 10CV lysis buffer (50mM HEPES pH 8, 500 mM NaCl, 5mM MgCl₂, 0.5mM TCEP, 10% glycerol, EDTA-free protease tablet)

Run the lysate through the column. Collect the flow through.

Wash the column with 30CV wash buffer (50mM HEPES pH 8, 500 mM NaCl, 5mM MgCl₂, 0.5mM TCEP, 10% glycerol, 20mM imidazole). Collect the wash fractions. Elute with an imidazole gradient of 100mM-500mM. Collect the elution fractions and analyze them by SDS PAGE.

Pool the fractions having the desired protein and buffer exchange the pool using a PD-10 desalting column (Cytiva #17085101) into the storage buffer (50mM HEPES pH 8, 150mM NaCl, 5mM MgCl₂, 0.5mM TCEP, 10% glycerol).

Seed 0.3 X 10⁶ cells onto collagen-coated coverslips. Once attached, starve the cells in media without serum or in low serum for 24h 0m 0s to induce ciliogenesis.

Set up an incubation chamber for ALOD4-mNeon staining: place a metal tray (covered with a layer of parafilm) on ice for 20-30 minutes before paraformaldehyde (PFA) fixation.

Aspirate media and fix cells in 4% PFA in 1X PBS for 0h 10m 0s at Room temperature.

Wash the cells three times in 1X PBS; transfer the coverslips to the incubation chamber and incubate them for 1h 0m 0s in 4μM ALOD4-mNeon diluted in 1% BSA 1X PBS, sterilized by passage through a 0.2 μm filter.

Wash the cells twice in 1X PBS; fix the cells again in 2% PFA diluted in 1X PBS for 5 minutes On ice. Wash the cells again twice in 1X PBS.

Optional: stain with DAPI (1:1000) for 4 minutes on ice. Wash the cells twice in 1X PBS and mount them onto clean glass slides using 4 µl Mowiol. Air-dry the coverslips and proceed to imaging.