TITLE: Golgi immunopurification (Golgi-IP) for subcellular metabolite 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 metabolite 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 metabolomics 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 other medium or treatments at this step. Incubation time will depend on your application.

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 293 T cells, other cells need to be optimized) gently on ice and avoid making bubbles. 
 


SECTION: Homogenization and Golgi-IP
19. Use 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 polar metabolite samples
26. Resuspend the IP samples in 50 µL of freezing cold 80 % (v/v) MeOH with isotopically labeled amino acids (500 nM) as internal standards.
 




SECTION: Processing of polar metabolite samples
27. Place samples in ice and start Golgi-IP for the next one (remember you are on a strict timed schedule).
SECTION: Processing of polar metabolite samples
28. At this point you should have WC  samples (25 µL from step 17 ) in the 1.5 mL tube ➂ and IP samples (50 µL from step 26) in the 1.5 mL tube ➄ with beads still in it.   

SECTION: Processing of polar metabolite samples
29. After 10 min  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 polar metabolite samples
30. For WC samples, add 225 µL freezing cold 80 % (v/v) MeOH with isotopically labeled amino acids to  tube ➂. Then vortex briefly.
SECTION: Processing of polar metabolite samples
31. For WC and Golgi-IP samples,  centrifuge  at top speed (15000 rpm, 15 min, 4 °C) and transfer the supernatant to a set of new tubes. Store WC an IP samples in these new tubes (from step 30) at -80 °C. On the day of LC/MS measurement, vortex samples for 10 min  at 4 °C and  centrifuge  at top speed (15000 rpm, 15 min, 4 °C). Then transfer supernatant to autosampler vials.