Immunoblotting of macrophages and microglia
Narayana Yadavalli, Shawn M. Ferguson
Abstract
This protocol describes the preparation from cell lysate from cultured cells and immunoblotting procedure.
Attachments
Steps
Cell culture and treatments
Supplement RIPA buffer with Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor (Roche) and chill On ice.
Aspirate media from cells and rinse cells with PBS On ice. Aspirate PBS thoroughly.
Pipette RIPA lysis buffer onto cells and scrape cells using a cell lifter (Corning).
Pipette lysis buffer containing cell mass into Eppendorf tube.
Treat with 25 units of benzinase for 0h 5m 0s.
Spin down at 14000rpm,4°C.
Collect supernatant.
Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).
Prepare samples at desired concentration and add 4x Laemmli buffer.
Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
Incubate samples at 95°C for 0h 3m 0s.
During this incubation, prepare gel apparatus with Mini PROTEAN TGX 4-15% tris-glycine gels (Bio-Rad) and running buffer.
Load samples into gel and run until dye front reaches bottom (250 V).
Remove gel and set up transfer cassette with nitrocellulose membrane.
Transfer at 100 V for 1h 0m 0s in tris-glycine transfer buffer.
Remove nitrocellulose membrane and stain for total protein with ponceau stain.
Wash with milliQ water.
Block membrane with 5% milk in TBST for 1h 0m 0s at Room temperature.
Incubate membrane with primary antibodies in 2.5% milk in TBST 1h 0m 0s at 4°C.
Wash membrane.
Wash membrane for 0h 10m 0s with TBST. (1/3)
Wash membrane for 0h 10m 0s with TBST. (2/3)
Wash membrane for 0h 10m 0s with TBST. (3/3)
Incubate membrane with secondary antibodies conjugated HRP for 1h 0m 0s.
Wash membrane.
Wash membrane for 0h 5m 0s with TBST. (1/3)
Wash membrane for 0h 5m 0s with TBST. (2/3)
Wash membrane for 0h 5m 0s with TBST. (3/3)
Image membranes using a Biorad Chemidco.
