TITLE: Organelle isolation from mouse tissues expressing organelle tags
AUTHORS: Rotimi Fasimoye, Emily Dickie, Matthew Taylor, Francesca Tonelli, Dario R Alessi

[DESCRIPTION]
We describe here a method to perform the rapid isolation of intact lysosomes from mouse tissues expressing a lysosome-localized TMEM192-3×HA fusion protein (“LysoTag”) (as in JAX stock #035401, RRID:IMSR_JAX:035401). The fusion protein marks lysosomes with a triple HA epitope, enabling the rapid isolation of intact tagged organelles from tissues using an anti-HA antibody. The organelles purified using this method are highly enriched, intact, largely contaminant-free and can be used for various downstream applications, including immunoblotting analysis and proteomic analysis (as described in dx.doi.org/10.17504/protocols.io.ewov1o627lr2/v1), but also lipidomic or metabolomic analysis (as described in dx.doi.org/10.17504/protocols.io.bybjpskn).
This method can be adapted to isolate other tagged organelles from tissues.


[STEPS]
SECTION: 1) Anti-HA Magnetic beads preparation
1. 1.1) Transfer n x 100 µl of anti-HA Magnetic Beads (where n = number of samples) into a low binding Eppendorf tube on ice.
SECTION: 1) Anti-HA Magnetic beads preparation
2. 1.2) Immobilize the beads by placing the tube into a Dyna-Mag tube holder for 30s.
SECTION: 1) Anti-HA Magnetic beads preparation
3. 1.3) Remove the supernatant using a pipette.
SECTION: 1) Anti-HA Magnetic beads preparation
4. 1.4) Gently resuspend the beads in 1ml of KPBS.
SECTION: 1) Anti-HA Magnetic beads preparation
5. 1.5) Repeat steps 1.2 to 1.4.
SECTION: 1) Anti-HA Magnetic beads preparation
6. 1.6) Immobilize the beads by placing the tube into a Dyna-Mag tube holder for 30s.
SECTION: 1) Anti-HA Magnetic beads preparation
7. 1.7) Remove the supernatant using a pipette.
SECTION: 1) Anti-HA Magnetic beads preparation
8. 1.8)	Gently resuspend the beads from step 1.7 in n x 100 µl of KPBS (where n = number of samples) to make a 1:1 slurry.
SECTION: 1) Anti-HA Magnetic beads preparation
9. 1.9) Aliquot the washed beads from step 1.8 into fresh low-binding Eppendorf tubes (100 μl of slurry for each sample).
SECTION: 1) Anti-HA Magnetic beads preparation
10. 1.10) Leave the tubes on ice until use (step 3.1).
SECTION: 2) Tissue homogenisation
11. 2.1) Collect tissues from mice culled by cervical dislocation.
Note: We recommend collecting and processing tissues from one mouse at a time.

SECTION: 2) Tissue homogenisation
12. 2.2) Rinse the tissues briefly in cold PBS.

SECTION: 2) Tissue homogenisation
13. 2.3) Transfer the tissues to a Falcon tube containing cold PBS on ice.
SECTION: 2) Tissue homogenisation
14. Note: The following steps should be performed in a cold room.
2.4) Transfer each tissue into a 2 ml Dounce homogeniser pre-washed with PBS.
Lung: Process the whole organ.
Brain: Process one hemisphere.
Other tissues might need optimisation.

SECTION: 2) Tissue homogenisation
15. 2.5) Add 1 ml of ice cold supplemented KPBS to each tissue sample.
SECTION: 2) Tissue homogenisation
16. 2.6) Homogenise the tissues as follows:
Lung: homogenise with 25 strokes.
Brain: homogenise with 25 strokes.
Other tissues might need optimisation.
Note: We recommend performing the first 5 strokes of lung homogenisation before adding the KPBS, as residual air in the lungs can make homogenisation difficult.

SECTION: 2) Tissue homogenisation
17. 2.7) Transfer the homogenate from step 2.6 to a low-binding Eppendorf tube on ice using a P1000 pipette.
Note: The pipette tip can be cut to ensure retrieval of all material.

SECTION: 2) Tissue homogenisation
18. 2.8) Centrifuge at 1000 g for 2 min at 4°C to remove debris.
SECTION: 2) Tissue homogenisation
19. 2.9) Transfer the supernatant from Step 2.8 into a new low-binding Eppendorf tube on ice. 
SECTION: 2) Tissue homogenisation
20. 2.10) For each sample, transfer 25-50 μL to a new low binding Eppendorf (= input) and keep on ice.
Note: 
Rinse the Dounce homogeniser with a generous amount of MilliQ water 3 times and cold PBS once between samples.
Proceed to organelle isolation as rapidly as possible after tissue homogenisation to avoid organelle degradation.


SECTION: 3) Organelle isolation
21. 3.1) Add the remaining homogenate from step 2.10 to the pre-washed HA-Magnetic beads (Step 1.10).
SECTION: 3) Organelle isolation
22. 3.2) Mix gently by flicking the bottom of the tube. Disperse any clumps by gently pipetting up and down 5 times.
SECTION: 3) Organelle isolation
23. 3.3) Incubate with agitation on a Belly Dancer orbital shaker for 5 minutes at 4°C.
SECTION: 3) Organelle isolation
24. The following steps should ideally be performed in a cold room. If not available, keep working on ice.

3.4) Place the tubes from Step 3.3 on a magnetic tube holder for 30s to immobilise the beads.
SECTION: 3) Organelle isolation
25. 3.5) Discard the supernatant (a sample can be collected and considered as a flow-through).
SECTION: 3) Organelle isolation
26. 3.6) Resuspend the beads from Step 3.5 in 1 ml of supplemented KBPS.
SECTION: 3) Organelle isolation
27. 3.7) Immobilise the beads by placing the tubes in a Dyna-Mag tube holder for 30s.
SECTION: 3) Organelle isolation
28. 3.8) Discard the supernatant.
SECTION: 3) Organelle isolation
29. 3.9) Repeat steps 3.6 to 3.8.
SECTION: 3) Organelle isolation
30. 3.10) Resuspend the beads in 1 ml of supplemented KPBS and transfer to a new low binding Eppendorf tube on ice.
SECTION: 3) Organelle isolation
31. 3.11) Place the tubes in a Dyna-Mag tube holder for 30s.
SECTION: 3) Organelle isolation
32. 3.12) Discard the supernatant.

The organelle IP beads (from step 3.12) and the input (from step 2.10) can now be processed for either immunoblotting analysis or proteomic analysis (as described in dx.doi.org/10.17504/protocols.io.ewov1o627lr2/v1), or for lipidomic or metabolomic analysis (as described in dx.doi.org/10.17504/protocols.io.bybjpskn).
Note: If isolating organelles for subsequent lipidomic or metabolomic analysis, use KPBS (instead of supplemented KPBS) in steps 3.6-3.10.
The input (Step 2.10) and organelle IP beads (Step 3.12) samples can be stored directly at -80°C and processed for analysis at a later time point. This allows the collection and processing of tissues from all mice in the same experimental cohort before subsequent analysis.