Lysosome immunoprecipitation in C. elegans

Procedure is done on the bench on ice.

Prepare the beads by washing the total amount three times with cold KBPS, using a magnet, and aliquoting them into the 2 mL tubes. Each IP needs a 160 ul of beads.

1. Grow ~160,000 worms (10 * 15 cm plates) to day1 adults.
2. Wash 3X in M9.
3. Wash 1X in cold KPBS.
4. Transfer worms in 2 ml KPBS into a Dounce homogenizer on ice.
5. Homogenize sample with 100+ strokes on ice. Depending on the homogenizer may take significantly more than 10 strokes to break the worm (it usually take 50*6 strokes with our homogenizer). We suggest proceeding in increments of 50 strokes with breaks in between to allow for visual assessment of the progress and avoid excessive heat generation.
6. Transfer sample into a 2 mL centrifuge tube. Centrifuge at 1000 g for 3 min at 4 ˚C.
7. Put the remaining sup (it has the organelles) on the BEADS and resuspend (May also save the pellet (containing nuclei and unbroken cells).
8. Rotate in 4 ˚C for 6 mins (Rotate in 20 ˚C  would increase binding efficiency) (everything from now on is in the cold room).
9. Put on magnet, then separate beads and sup/flowthrough (May also save the sup/flowthrough (containing cytosol and other organelles) as control).
10. Wash the bound bead fraction 4 times with 2 ml cold KPBS (only by few pipetings).
11. Aspirate all KPBS. Save the remaining BEADS.

KPBS:

KCl                 136 mM  74.5513 g·mol⁻¹ 10.14g for 1L
KH2PO4         10 mM  136.09 g mol-1 1.36g for 1L

*pH using KOH or HCl, not NaOH because we don’t want Na+ ions

*Filter through 0.22 um filter, store at 4°C

*Prepare using LC/MS grade water

*pH 7.25

Reagents

Anti-HA magnetic beads (Thermo Fisher Scientific, cat. no. 88837)