TITLE: Introducing GolgiTAG to Cells and Immunoprecipitation of Golgi
AUTHORS: Rotimi Y. Fasimoye, Dario R. Alessi

[DESCRIPTION]
Golgi apparatus is essential to the secretory pathway of the cell and by extension helps to maintain cellular homeostasis. It is the main transportation hub of the cell, where molecules destined for other organelles within the cytoplasm or outside the cell for secretion are packaged into vesicles. The purification of Golgi using available techniques is time-consuming and laborious, as it requires various centrifugation steps, sometimes in gradient buffers, which reduce yield, compromise its integrity, and increase the chance of contamination, especially from endoplasmic reticulum. Furthermore, depending on the cell type or tissue, the methods for Golgi purification differs. Here, we present a protocol for rapidly purifying Golgi from the mammalian HEK293 cell line, using high affinity anti-HA magnetic beads. This method relies on the immunoprecipitation, in phosphate buffer, of HA-tagged integral membrane protein of the Golgi complex, TMEM115. The HA-tagged TMEM115 expressing vector is packaged into a lentivirus, therefore various mammalian cell lines can be transduced, giving stable expression levels. Our protocol is fast, approximately 10 min, and can be used on various cell lines and tissues without any modification. The Golgi purified using this method are highly enriched, intact, contaminant-free and, depending on solubilisation buffer, could be used for various downstream applications, such as proteomics and metabolomics.

[STEPS]
SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
1. 
 

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
2. Grow 2 dishes of HEK293FT cells to 50-60% confluency in Growth media in 10cm Petri Dish.
 

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
3. Prepare a transfection mix to generate a lentivirus expressing the GolgiTag in 1.5ml Eppendorf tube containing:
3.8 µg
2.2 µg 
6 µg 
300 µL  
 

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
4. Prepare a transfection mix to generate a lentivirus vector expressing ControlTag in 1.5ml Eppendorf tube containing:
3.8 µg
2.2 µg 
6 µg 
300 µL  
 

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
5. Prepare PEI mixture in 1.5ml Eppendorf tube. 
40 µL  dissolved in distilled water 
600 µL
SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
6. Incubate each mixture (Step 3, 4, 5) separately for ~5 min at Room temperature.

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
7. Add 310 µL to the GolgiTag in 1.5ml Eppendorf tube (Step 3) and ControlTag in 1.5ml Eppendorf tube (Step 4) mixtures.

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
8. Mix and incubate each mixture at Room temperature for 30 min.

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
9. Add each mixture dropwise using a P1000 sterile pipette into a single HEK293FT containing plate.
SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
10. Incubate cells 37 °C for 1440 min.

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
11. Replace media with fresh Growth Media (discard old media) and incubate cells at 37 °C for further  1440 min.

SECTION: Packaging GolgiTag and ControlTag construct into lentivirus system
12. Collect media that contains the lentivirus and pass through 0.45µm syringe filter. This is now the lentivirus infection media. This could be used immediately or snap frozen in liquid nitrogen and stored at -80 °C.

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
13. 
 

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
14. Mix 5 mL produced in step 12 with 5 mL (in a 15ml Eppendorf tube).

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
15. Add Polybrene reagent to Mix produced in step 14 using a stock of 10mg/ml dissolved in MilliQ water (which had been sterile filtered) to a final concentration of 10 microgram per milliliter (μg/mL).

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
16. Gently add media to 10cm plate of HEK293 cells already at ~60% confluency.
 

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
17. Incubate at 37 °C for 1440 min.

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
18. Change media to Growth Media and incubate 37 °C for another 1440 min.

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
19. To select for cells stably expressing GolgiTag or ControlTag, replace media with 10 mL.

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
20. After 1440 min, there will be observable death and a considerable number of dead floating cells will be observed.

SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
21. Change Selection Media each 24h for 3-5 days (depending on the efficiency of the transduction process). At this stage colonies of cells stably expressing the GolgiTag or empty vector should have reached 100% confluency. 
SECTION: Infecting (Transducing) and Selecting cells stably expressing GolgiTag
22. Split cells into more dishes maintaining cells in Selection Media. 
 

SECTION: Cell preparation for GolgiTag and ControlTag immunoprecipitation (To be undertaken 1-2 days prior to immunoprecipitation)
23. Split cells stably expressing GolgiTag or ControlTag in 15cm Petri dish. Allow to grow near confluency (approximately 24-48h depending on the seeding density).
SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
24. 
 

SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
25. Pipette 200 µL into 1.5ml Eppendorf tube.

SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
26. Insert tube containing bead anti-HA bead slurry onto a DynaMag™ Spin Magnet for 30 s and carefully remove supernatant using a P1000 pipette.

SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
27. Remove bead from DynaMag™ Spin Magnet, add 1 mL, gently pipette up and down to disperse any clumps, put on DynaMag™ Spin Magnet for 30 s and carefully remove supernatant using a P1000 pipette.

SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
28. Repeat wash step described in step 27 a further 3 times:
SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
28.1. (Wash 1/3): Remove bead from DynaMag™ Spin Magnet, add 1 mL, gently pipette up and down to disperse any clumps, put on DynaMag™ Spin Magnet for 30 s and carefully remove supernatant using a P1000 pipette.
SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
28.2. (Wash 2/3): Remove bead from DynaMag™ Spin Magnet, add 1 mL, gently pipette up and down to disperse any clumps, put on DynaMag™ Spin Magnet for 30 s and carefully remove supernatant using a P1000 pipette.
SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
28.3. (Wash 3/3): Remove bead from DynaMag™ Spin Magnet, add 1 mL, gently pipette up and down to disperse any clumps, put on DynaMag™ Spin Magnet for 30 s and carefully remove supernatant using a P1000 pipette.
SECTION: Pre-clearing of anti-HA beads (To be done on the day of immunoprecipitation before cell lysis)
29. After the last wash step, incubate the bead slurry in 50 µL and keep beads on ice.

SECTION: Cell lysis for immunoprecipitation
30. 
 

SECTION: Cell lysis for immunoprecipitation
31. Place cells on ice slighted tilted to enable medium to be easily aspirated.

SECTION: Cell lysis for immunoprecipitation
32. Remove media and immediately wash plates with 10 mL.

SECTION: Cell lysis for immunoprecipitation
33. Immediately, gently aspirate PBS from cells.
SECTION: Cell lysis for immunoprecipitation
34. Gently wash one more time repeating steps 32 and 33:
SECTION: Cell lysis for immunoprecipitation
34.1. Remove media and immediately wash plates with 10 mL.

SECTION: Cell lysis for immunoprecipitation
34.2. Immediately, gently aspirate PBS from cells.
SECTION: Cell lysis for immunoprecipitation
35. Carefully remove PBS and add 1 mL.
SECTION: Cell lysis for immunoprecipitation
36. Whilst cells are maintained on ice, scrape cells off dish with a disposable cell lifter.

SECTION: Cell lysis for immunoprecipitation
37. Carefully transfer scrapped cells using an P1000 pipette into a 1.5ml Eppendorf tube.
SECTION: Cell lysis for immunoprecipitation
38. Pellet cell by centrifugation at 1000 x g, 2 min, 4 °C.

SECTION: Cell lysis for immunoprecipitation
39. Carefully discard supernatant using an P1000 Pipette.
SECTION: Cell lysis for immunoprecipitation
40. Resuspend pellet in 950 µL.

SECTION: Cell lysis for immunoprecipitation
41. Pipette cells up and down with a P1000 pipette ensure cells are in suspension and remove 50 µL into a 1.5 ml Eppendorf tube marked as “cell lysate INPUT”. Keep this on ice 
Place the tube containing the remaining ~900µl of resuspended cells on ice and take sample in the cold room.
 

SECTION: Cell lysis for immunoprecipitation
42. Pipette the 900 µL into a clean 2ml Dounce homogeniser.

SECTION: Cell lysis for immunoprecipitation
43. Homogenise cells with exactly 25 strokes, making sure bubbles are avoided. 
To verify efficacy of homogenisation, take 10µl on a slide, briefly check under the microscope. There should be about at least 80% cell lysis, indicated by the visible presence of nuclei without surrounding cytoplasm. 
 

SECTION: Cell lysis for immunoprecipitation
44. Centrifuge lysate at 1000 x g, 2 min, 4 °C. This is to remove any non-lysed cells, nuclei and debris such as cell membranes.
SECTION: Cell lysis for immunoprecipitation
45. Remove supernatant (which should contain organelles) into a new 1.5 ml Eppendorf tube. The lysate at this stage will be between 700-800µl.
SECTION: Cell lysis for immunoprecipitation
46. Remove 50 µL from the homogenate into a separate 1.5ml Eppendorf labelled as IP INPUT. Keep on ice.

SECTION: GolgiTag and ControlTag immunoprecipitation
47. 
 

SECTION: GolgiTag and ControlTag immunoprecipitation
48. Take out Eppendorf tube containing the washed anti-HA beads from step 29.
SECTION: GolgiTag and ControlTag immunoprecipitation
49. Add the cell homogenate from step 45 into the eppendorf tube containing the HA-beads.
SECTION: GolgiTag and ControlTag immunoprecipitation
50. Ensure that bead clumps are dispersed by gently pipetting up and down 5 times using a P1000 Pipette.
SECTION: GolgiTag and ControlTag immunoprecipitation
51. Incubate the cell homogenate-HA-bead slurry for 5 min on orbiter. 

SECTION: GolgiTag and ControlTag immunoprecipitation
52. Separate bead by putting tube on DynaMag™ Spin Magnet for 30 s.

SECTION: GolgiTag and ControlTag immunoprecipitation
53. Collect 50 µL of supernatant into separate 1.5ml Eppendorf tube label as FT Supernatant (FT = flow-through). Keep on ice.

SECTION: GolgiTag and ControlTag immunoprecipitation
54. Wash bead with 1000 µl of ice cold KPBS 3 times:
SECTION: GolgiTag and ControlTag immunoprecipitation
54.1. (Wash 1/3): Wash bead with 1000 µL.

SECTION: GolgiTag and ControlTag immunoprecipitation
54.2. (Wash 2/3): Wash bead with 1000 µL.

SECTION: GolgiTag and ControlTag immunoprecipitation
54.3. (Wash 3/3): Wash bead with 1000 µL.

SECTION: GolgiTag and ControlTag immunoprecipitation
55. Incubate beads with 500 µL and prepare a slurry by pipetting up and down 5 times with a P1000 pipette and transfer slurry into a clean 1.5 ml Eppendorf tube.
 


SECTION: GolgiTag and ControlTag immunoprecipitation
56. Insert tube containing anti-HA bead slurry onto a DynaMag™ Spin Magnet for 30 s and carefully remove supernatant using a P1000 pipette.

SECTION: GolgiTag and ControlTag immunoprecipitation
57. Add 80 µL to the HA beads containing the immunoprecipitated Golgi.
 

SECTION: GolgiTag and ControlTag immunoprecipitation
58. Incubate for 10 min on ice.

SECTION: GolgiTag and ControlTag immunoprecipitation
59. Place tube on DynaMag™ Spin Magnet for 30 s to separate beads.

SECTION: GolgiTag and ControlTag immunoprecipitation
60. Collect supernatant into new Eppendorf tube.
 

SECTION: GolgiTag and ControlTag immunoprecipitation
61. To ensure total removal of beads, add a further 20 µL to HA-Beads and place the beads again on DynaMag™ Spin Magnet for 30 s and collect supernatant into Eppendorf tube containing the solubilized Golgi in step 60.

SECTION: GolgiTag and ControlTag immunoprecipitation
62. Determine the protein concentration of the IP using a BCA Protein Assay Kit. Use 5 µL (do not dilute) for quantification purpose. Quantification should be done in duplicate.

SECTION: GolgiTag and ControlTag immunoprecipitation
63. Depending on future application samples can be snap frozen in liquid nitrogen and stored at -80 °C or diluted into 4xLDS loading buffer supplemented with fresh 5% (by vol) 2-mercaptoethanol prior to analysis on SDS-polyacrylamide gel electrophoresis and immunoblot analysis. For Immunoblot purpose, 2-4 µg of solubilised Golgi is enough to detect Golgi markers (GM130, ACBD3 and GOLGIN97).

SECTION: Preparation of INPUT sample
64. Pellet cells by centrifugation at 1000 x g, 2 min, 4 °C.

SECTION: Preparation of INPUT sample
65. Discard supernatant and resuspend cell pellet in 80 µL.

SECTION: Preparation of INPUT sample
66. Pipette up and down 10-20 times with a P200 pipette.
SECTION: Preparation of INPUT sample
67. Incubate lysate on ice for 20 min.

SECTION: Preparation of INPUT sample
68. Centrifuge lysate at 13000 x g, 10 min, 4 °C.

SECTION: Preparation of INPUT sample
69. Collect clarified cell lysate supernatant in fresh tube and determine protein concentration using a BCA Protein Assay Kit. Dilute an aliquot of the supernatant in lysis buffer at a ratio 1:10, before protein quantification.
SECTION: Preparation of INPUT sample
70. Depending on future application the clarified cell lysate can be snap frozen in liquid nitrogen and stored at -80 °C or diluted 4X into 4xLDS loading buffer supplemented with fresh 5% (by vol) 2-mercaptoethanol prior to analysis on SDS-polyacrylamide gel electrophoresis and immunoblot analysis.

SECTION: Preparation of Flow-through and IP INPUT sample
71. Add 100 µL to tubes marked FT and IP INPUT.

SECTION: Preparation of Flow-through and IP INPUT sample
72. Mix thoroughly by pipetting up and down using P1000.
SECTION: Preparation of Flow-through and IP INPUT sample
73. Incubate on ice for 20 min.

SECTION: Preparation of Flow-through and IP INPUT sample
74. Centrifuge lysate at 13000 x g, 10 min, 4 °C.

SECTION: Preparation of Flow-through and IP INPUT sample
75. Collect clarified cell lysate supernatant in fresh tube and determine protein concentration using the BCA method. Dilute an aliquot of the supernatant in lysis buffer at a ratio 1:10, before protein quantification.
SECTION: Preparation of Flow-through and IP INPUT sample
76. Depending on future application the clarified cell lysate can be snap frozen in liquid nitrogen and stored at -80 °C or diluted 4X into 4xLDS loading buffer supplemented with fresh 5% (by vol) 2-mercaptoethanol prior to analysis on SDS-polyacrylamide gel electrophoresis and immunoblot analysis.