Lysosome isolation and analysis

Lysosome Isolation and Analysis

Note: This protocol was prepared based on the manual of Lysosome Purification Kit from Biovision (K235-50).

1.      Pellet 2 x10⁷ cells by centrifugation at 600 x g for 10 min.

2.      Remove and discard the supernatant.

3.      Add 500 μl of lysosome isolation buffer (K235-50-1, Biovision) to resuspend the cells and then vortex for 5 seconds, put on ice for 2 min.

4.      Homogenize the cells with a 3 mL precooled Glass Grinder (Kontes), stroke the sample for 60 times on ice.

5.      Transfer the homogenate to a fresh 1.5 mL Eppendorf tube.

6.      Add 500 μl of lysosome enrichment buffer (K235-50-2, Biovision) to the homogenate, invert the tube 10 times to mix.

7.      Centrifuge at 500 x g for 10 min at 4 °C.

8.      Transfer the supernatant (~900 μl) to a separate 1.5 mL Eppendorf tube and keep on ice.

9.      Prepare five gradient solutions (#1 - #5, gradient#1 and #5 represent the top and bottom layers of the gradient respectively) with lysosome gradient (K235-50-3) and lysosome enrichment buffer in five centrifuge tubes. Make sure to mix enough gradients for the number of samples to be assayed. For a 5 mL gradient extraction, prepare 800 μl of every gradient containing:

10.  Add protease inhibitor cocktail (K235-50-4) at a ratio of 1:1000 (1 μl of protease inhibitor to 1 mL gradient solutions in step 9).

11.  Make a discontinuous density gradient by carefully adding the prepared lysosome gradient/lysosome enrichment gradient solutions (#1 - #5) to a 5mL open-top thinwall ultracentrifuge tube (326819, Beckman Coulter), start making the discontinuous gradient by adding gradient #5, do not shake or move the tubes during the preparing process.

12.  Dilute the prepared cell lysate from step 8 by mix 1 Part of lysosome gradient (~300 μl) with 3 Parts of cell lysate (~900 μl).

13.  Carefully add the diluted cell lysate to the top of the prepared discontinuous density gradient.

14.  Put the ultracentrifuge tube into a bucket of SW 55 Ti Rotor (Beckman Coulter).

15.  Centrifuge the tubes using ultracentrifuge (XL-70, Beckman Coulter) for 2 hrs at 145,000 x g at 4 °C

16.  Carefully withdraw the lysosome fraction band, which is visible on the top 1/10^th mL of the gradient volume, by using an extra-long pipette tip starting from top of the gradient.

17.  Purify the lysosomes by mix the collected lysosome fraction with 2 volume of PBS.

18.  Vortex gently and then centrifuge for 30 min at 18,000 x g at 4 °C.

19.  Discard the supernatant and keep the pellet containing the purified lysosomes.

20.   For activity assay, resuspend the pellet with PBS and measure the protein concentration; for long term storage purpose, resuspend in PBS, aliquot and snap freeze in liquid nitrogen and keep the frozen lysosomes at -80 °C; for western blot analysis, resuspend the pellet with RIPA buffer, determine the protein concentration and subject to SDS-PAGE.