TITLE: Golgi immunopurification (Golgi-IP) for subcellular lipid profiling
AUTHORS: Wentao Dong, Eshaan S Rawat, Monther Abu-Remaileh

[DESCRIPTION]
The Golgi is a membrane-bound organelle that is central to protein and lipid processing, sorting and secretion in the cell. Despite its critical cellular function, there has been challenges to quantitatively assess Golgi lipid profiles. To overcome this hurdle, we developed a rapid harvesting and purification method using immunoprecipitation (Golgi-IP). This protocol provides details for preparing Golgi-IP lipidomics samples.

[STEPS]
SECTION: Preparation of homogenizers and sample tubes
1. Wash the glass vessel homogenizer with MilliQ Water, 10 times each. Wash the tissue grinder homogenizer thoroughly with DI Water and MilliQ Water, especially the gap between the white parts, don’t touch the part that goes into the glass vessel. Then dry upside-down using paper towels. Carefully place the glass vessels against something to prevent falling down. Minimize any contact between the grinder and anything else.   
SECTION: Preparation of homogenizers and sample tubes
2. Prepare microcentrifuge tubes as follows on a metal rack on ice (for each sample, from left to right): ➀ 2 mL tube for cell suspension from harvesting; ➁ 1.5 mL tube for post-homogenization cell suspension (organelles in supernatant, membranes in pellet); ➂ 1.5 mL tube for whole cell sample; ➃ 1.5 mL tube for beads; ➄ 1.5 mL tube for post-magnetic samples; ➅ 1.5 mL tube for final Golgi-IP samples. Carefully label tubes ➂ and ➅ with detailed samples and experiments names.   
   

SECTION: Preparation of Anti-HA beads
3. Pool all required volumes together (100 µL/ plate, e.g. 800 uL total for 8 plates, extra is not needed).

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SECTION: Preparation of Anti-HA beads
4. Shake bottle very well before removing as beads tend to sink to the bottom.
SECTION: Preparation of Anti-HA beads
5. Wash 3 x with the same volume cold clean MS grade KPBS, after settling on magnet. Remove the holder from the magnet itself before dispending washing KPBS to avoid wetting the magnet.

SECTION: Preparation of Anti-HA beads
6. Resuspend with KPBS with same amount of volume originally removed from bottle.
SECTION: Preparation of Anti-HA beads
7. Aliquot 100 µL into each 1.5 mL labeled tubes ➃.

SECTION: Cell preparation before harvesting
8. Wash the first set of 15cm plates (each set has two plates) with 10 mL of DMEM/plate (for HEKS, use no serum + no antibiotics).

SECTION: Cell preparation before harvesting
9. Replace with 10 mL of DMEM/plate for an hour. You can also use full media or other treatments based on your experimental needs.

SECTION: Cell preparation before harvesting
10. 10 mL The second set of plates will be washed 20 min later after the first set and so on. 
 



SECTION: Cell preparation before harvesting
11. One hour after DMEM wash, take the first set of plates from incubator to bench and place on ice.   
SECTION: Cell preparation before harvesting
12. Decant the media.  Then Wash the cells twice by pouring ~5 mL cold clean MS grade PBS on the edge of the plate, decant the first time and then aspirate the second time. 

SECTION: Cell harvesting
13. Add 950 µL of cold KPBS to each 15-cm dish.   

SECTION: Cell harvesting
14. Scrape the cells down to the bottom of your plates with a cell lifter and transfer the cell suspension into the 2ml tube ➀.  Note: this step should be carefully accounted for and done the same between plates. Visually check (with an angle) that all cells have been harvested. We are using a 2mL tube since 950 uL KPBS + cells gives around 2mL volume.
SECTION: Cell harvesting
15. Spin at 1000 x g for 2 min at 4 °C.
 





SECTION: Cell harvesting
16. Aspirate the supernatant and resuspend the pellets with 950 µL  cold KPBS.   

SECTION: Cell harvesting
17. From this resuspended sample, take 25 µL for whole cell in the 1.5 mL tube ➂.   
Note: if pellet mixer is used instead of douncer, resuspend the pellets with 100 µL  cold KPBS in step 16, homogenize cells and then replenish to 950 uL and follow step 17. 
SECTION: Homogenization and Golgi-IP
18. Transfer the remainder (925 µL) of cells into a clean and pre-chill douncer. Dounce the cells 25 times (for HEK293T cells, other cells need to be optimized) gently on ice and avoid making bubbles. 
 


SECTION: Homogenization and Golgi-IP
19. Use 2 mL serological pipet to transfer sample from douncer into the 1.5 mL tubes ➁.   
SECTION: Homogenization and Golgi-IP
20. Spin 1,000g for 2 min at 4 °C.

SECTION: Homogenization and Golgi-IP
21. a.      Wash douncers during this spin for subsequent harvesting   
SECTION: Homogenization and Golgi-IP
22. Put the remaining supernatant (it contains the organelles) on the 1.5 ml tube ➃ with beads and resuspend by pipetting up and down ONE TIME.
 


SECTION: Homogenization and Golgi-IP
23. Rock in cold room for 3 min (everything from now on is in the cold room). 
 



SECTION: Homogenization and Golgi-IP
24. Put the ➃ tube on magnet. Count at least 25 s to allow for beads to be pulled by magnets. 
 


SECTION: Homogenization and Golgi-IP
25. Wash the bound fraction 3 times with 1 mLcold KPBS. Then aspirate all cold KPBS.
 


SECTION: Processing of nonpolar lipids samples
26. For nonpolar metabolites (lipidomics), both IP and WC samples, resuspend in 1000 µL  of chloroform:methanol at ratio of 2:1 (v/v) with 1000x diluted Splashmix (Avanti). Then incubate for 10 min .

SECTION: Processing of nonpolar lipids samples
27. After 10 min  of finishing the last IP,  place IP samples in the tube ➄ on the magnet, collect supernatant, and transfer to the 1.5 mL tube ➅.   
SECTION: Processing of nonpolar lipids samples
28. For both WC and Golgi-IP samples, vortex for 60 min  in cold room. Then add 200 µL  of 0.9% (w/v) saline (VWR) and vortex for another 10 min in cold room. The mixture was centrifuged at 3000 x g for 5 min  at 4 °C . Then discard the top layer (MeOH and saline polar phase) and use bubbeling method to retrieve 600 µL from the bottom layer (chloroform containing lipids) to clean prechilled eppendorf tubes. Next speedvac the chloroform samples until dried. Then store lipidomics WC and IP samples at -80 °C. On the day of analysis, dried lipid extracts were reconstituted in 50 µL  of ACN:IPA:water 13:6:1 (v/v/v) and vortexed for 10 min  at 4 °C . Then samples are centrifuged for 15 min  at 4 °C at max speed, and then 45 µL  of supernatant is transferred into glass insert vials for LC/MS.