TITLE: Immunoblotting of macrophages and microglia
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes the preparation from cell lysate from cultured cells and immunoblotting procedure.

[STEPS]
SECTION: Cell culture and treatments
1. Supplement RIPA buffer with Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor (Roche) and chill on ice.

SECTION: Cell culture and treatments
2. Aspirate media from cells and rinse cells with PBS on ice. Aspirate PBS thoroughly.

SECTION: Cell culture and treatments
3. Pipette RIPA lysis buffer onto cells and scrape cells using a cell lifter (Corning).
SECTION: Cell culture and treatments
4. Pipette lysis buffer containing cell mass into Eppendorf tube.
SECTION: Cell culture and treatments
5. Treat with 25 units of benzinase for 5 min.

SECTION: Cell culture and treatments
6. Spin down at 14000 rpm, 5 min, 4 °C.

SECTION: Cell culture and treatments
7. Collect supernatant.
SECTION: Cell culture and treatments
8. Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).
SECTION: Cell culture and treatments
9. Prepare samples at desired concentration and add 4x Laemmli buffer.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
10. Incubate samples at 95 °C for 3 min.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
11. During this incubation, prepare gel apparatus with Mini PROTEAN TGX 4-15% tris-glycine gels (Bio-Rad) and running buffer.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
12. Load samples into gel and run until dye front reaches bottom (250 V).
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
13. Remove gel and set up transfer cassette with nitrocellulose membrane.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
14. Transfer at 100 V for 60 min in tris-glycine transfer buffer.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
15. Remove nitrocellulose membrane and stain for total protein with ponceau stain.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
16. Wash with milliQ water.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
17. Block membrane with 5% milk in TBST for 60 min at Room temperature.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
18. 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)
19. Wash membrane.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
20. Incubate membrane with secondary antibodies conjugated HRP for 60 min.

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
21. Wash membrane.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
22. Image membranes using a Biorad Chemidco.
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
19.1. Wash membrane for 10 min with TBST. (1/3)

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
19.2. Wash membrane for 10 min with TBST. (2/3)
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
19.3. Wash membrane for 10 min with TBST. (3/3)
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
21.1. Wash membrane for 5 min with TBST. (1/3)

SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
21.2. Wash membrane for 5 min with TBST. (2/3)
SECTION: Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
21.3. Wash membrane for 5 min with TBST. (3/3)