TITLE: Immunoblotting
AUTHORS: Will Hancock-Cerutti, Pietro De Camilli

[DESCRIPTION]
This protocol describes collection of protein from cultured cells and immunoblotting, including immunoblotting of large proteins using a proprietary tris-acetate buffer system.

[STEPS]
SECTION: Cell culture and treatments
1. Culture the HeLa-M cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin, and 2 Milimolar (mM) L-glutamine (all from Gibco).

SECTION: Cell culture and treatments
2. For any given experiment, plate the cells at such density so as to be approximately 90% confluent at the time of lysis.
SECTION: Cell culture and treatments
3. For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol. Lyse the cells 4320 min after siRNA transfection.

SECTION: Cell culture and treatments
4. For experiments using cGAMP, transfect 8 μg/L of cGAMP using 6 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol.

SECTION: Cell culture and treatments
5. For experiments using herring testes (HT)-DNA, transfect 1 µg HT-DNA using 3 µL Lipofectamine 2000 (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol.

SECTION: Cell lysis and sample preparation
6. Supplement RIPA buffer with Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor (Roche) and chill on ice.

SECTION: Cell lysis and sample preparation
7. Aspirate media from cells and rinse cells with PBS on ice. Aspirate PBS thoroughly.

SECTION: Cell lysis and sample preparation
8. Pipette RIPA lysis buffer onto cells and scrape cells using a cell lifter (Corning).
SECTION: Cell lysis and sample preparation
9. Pipette lysis buffer containing cell mass into Eppendorf tube.
SECTION: Cell lysis and sample preparation
10. Incubate Eppendorf tube on ice for 30 min.

SECTION: Cell lysis and sample preparation
11. Every 10 minutes, pipette lysis mixture up and down 10 times with a P-200 pipette tip (a total of 3 cycles).
 

SECTION: Cell lysis and sample preparation
12. Centrifuge at 15000 x g for 10 min at 4 °C and collect the post-nuclear supernatant in a new Eppendorf tube.
 




SECTION: Cell lysis and sample preparation
13. Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).
SECTION: Cell lysis and sample preparation
14. Prepare samples at desired concentration and add 3x Laemmli buffer.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
15. Incubate samples at 95 °C for 5 min.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
16. During this incubation, prepare gel apparatus with Mini PROTEAN TGX 4-20% trisglycine gels (Bio-Rad) and running buffer.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
17. Load samples into gel and run until dye front reaches bottom (90-120 V).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
18. Remove gel and set up transfer cassette with nitrocellulose membrane.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
19. Transfer at 120 V for 60 min at 4 °C in tris-glycine transfer buffer.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
20. Remove nitrocellulose membrane and stain for total protein with ponceau stain.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
21. Wash with milliQ water.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
22. Block membrane with 5% milk in TBST for 60 min at 22 °C.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
23. Incubate membrane with primary antibodies in 2.5% milk in TBST 60 min at 4 °C.
 



SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
24. Wash membrane with TBST. Repeat a total of 3 times.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
25. Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 2.5% milk in TBST for 60 min at 22 °C.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
26. Wash membrane with TBST. Repeat a total of 3 times.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
27. Wash membrane with TBS. Repeat a total of 3 times.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
28. Image membranes using a Licor Odyssey Infrared Imager.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
24.1. Wash membrane for 5 min with TBST (1/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
24.2. Wash membrane for 5 min with TBST (2/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
24.3. Wash membrane for 5 min with TBST (3/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
26.1. Wash membrane for 5 min with TBST (1/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
26.2. Wash membrane for 5 min with TBST (2/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
26.3. Wash membrane for 5 min with TBST (3/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
27.1. Wash membrane for 5 min with TBS (1/3).

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
27.3. Wash membrane for 5 min with TBS (3/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
27.2. Wash membrane for 5 min with TBS (2/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
29. For VPS13C immunoblotting, lyse samples and collect the post-nuclear supernatant as above.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
30. Mix post-nuclear supernatant with NuPAGE LDS Sample Buffer and Reducing Agent (Thermofisher) and incubate for 10 min at 70 °C.

SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
31. During this incubation, prepare gel apparatus with NuPage Tris-Acetate 3-8% gels and NuPage Running Buffer (Thermofisher).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
32. Remove gel and set up transfer cassette with nitrocellulose membrane.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
33. Transfer at 0.05 mA for 960 min at 4 °C in NuPage transfer buffer (Thermofisher).

SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
34. Remove nitrocellulose membrane and stain for total protein with ponceau stain.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
35. Wash with milliQ water.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
36. Block membrane with 5% milk in TBST for 60 min at 22 °C.

SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
37. Incubate membrane with primary antibodies in 2.5% milk in TBST for 120 min at 22 °C.
 



SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
38. Wash membrane for with TBST. Repeat a total of 3 times.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
39. Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 2.5% milk in TBST for 60 min at 22 °C.

SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
40. Wash membrane with TBST. Repeat a total of 3 times.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
41. Wash membrane with TBS. Repeat a total of 3 times.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
42. Image membranes using a Licor Odyssey Infrared Imager.
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
38.1. Wash membrane for 5 min with TBST (1/3).

SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
38.3. Wash membrane for 5 min with TBST (3/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
38.2. Wash membrane for 5 min with TBST (2/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
40.1. Wash membrane for 5 min with TBST (1/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
40.2. Wash membrane for 5 min with TBST (2/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
40.3. Wash membrane for 5 min with TBST (3/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
41.1. Wash membrane for 5 min with TBS (1/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
41.3. Wash membrane for 5 min with TBS (3/3).
SECTION: Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
41.2. Wash membrane for 5 min with TBS (2/3).