Immunoblotting
Pietro De Camilli, Will Hancock-Cerutti
Abstract
This protocol describes collection of protein from cultured cells and immunoblotting, including immunoblotting of large proteins using a proprietary tris-acetate buffer system.
Attachments
Steps
Cell culture and treatments
Culture the HeLa-M cells at 37°C in 5% CO2 and DMEM containing 10% FBS, 100U/ml penicillin, 100mg/mL streptomycin, and 2millimolar (mM) L-glutamine (all from Gibco).
For any given experiment, plate the cells at such density so as to be approximately 90% confluent at the time of lysis.
For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol. Lyse the cells 72h 0m 0s after siRNA transfection.
For experiments using cGAMP, transfect 8μg/L of cGAMP using 6µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol.
For experiments using herring testes (HT)-DNA, transfect 1µg HT-DNA using 3µL Lipofectamine 2000 (ThermoFisher) in Opti-MEM (Gibco) per well according to manufacturer protocol.
Cell lysis and sample preparation
Supplement RIPA buffer with Protease Inhibitor Cocktail (Roche) and PhosStop phosphatase inhibitor (Roche) and chill 37On ice.
Aspirate media from cells and rinse cells with PBS 37On 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.
Incubate Eppendorf tube 37On ice for 0h 30m 0s.
Every 10 minutes, pipette lysis mixture up and down 10 times with a P-200 pipette tip (a total of 3 cycles).
Centrifuge at 15000x g,0h 0m 0s for 0h 10m 0s at 4°C and collect the post-nuclear supernatant in a new Eppendorf tube.
Determine protein concentration in sample using Pierce BCA assay (ThermoFisher).
Prepare samples at desired concentration and add 3x Laemmli buffer.
Gel electrophoresis and immunoblotting (Tris-glycine buffer system)
Incubate samples at 95°C for 0h 5m 0s.
During this incubation, prepare gel apparatus with Mini PROTEAN TGX 4-20% trisglycine gels (Bio-Rad) and running buffer.
Load samples into gel and run until dye front reaches bottom (90-120 V).
Remove gel and set up transfer cassette with nitrocellulose membrane.
Transfer at 120 V for 1h 0m 0s at 4°C 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 22°C.
Incubate membrane with primary antibodies in 2.5% milk in TBST 1h 0m 0s at 4°C.
Wash membrane with TBST. Repeat a total of 3 times.
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).
Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 2.5% milk in TBST for 1h 0m 0s at 22°C.
Wash membrane with TBST. Repeat a total of 3 times.
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).
Wash membrane with TBS. Repeat a total of 3 times.
Wash membrane for 0h 5m 0s with TBS (1/3).
Wash membrane for 0h 5m 0s with TBS (2/3).
Wash membrane for 0h 5m 0s with TBS (3/3).
Image membranes using a Licor Odyssey Infrared Imager.
Gel electrophoresis and immunoblotting (Tris-acetate buffer system)
For VPS13C immunoblotting, lyse samples and collect the post-nuclear supernatant as above.
Mix post-nuclear supernatant with NuPAGE LDS Sample Buffer and Reducing Agent (Thermofisher) and incubate for 0h 10m 0s at 70°C.
During this incubation, prepare gel apparatus with NuPage Tris-Acetate 3-8% gels and NuPage Running Buffer (Thermofisher).
Remove gel and set up transfer cassette with nitrocellulose membrane.
Transfer at 0.05 mA for 16h 0m 0s at 4°C in NuPage transfer buffer (Thermofisher).
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 22°C.
Incubate membrane with primary antibodies in 2.5% milk in TBST for 2h 0m 0s at 22°C.
Wash membrane for with TBST. Repeat a total of 3 times.
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).
Incubate membrane with secondary antibodies conjugated to IRdye 800CW or IRdye 680CW (1:10,000, Licor) in 2.5% milk in TBST for 1h 0m 0s at 22°C.
Wash membrane with TBST. Repeat a total of 3 times.
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).
Wash membrane with TBS. Repeat a total of 3 times.
Wash membrane for 0h 5m 0s with TBS (1/3).
Wash membrane for 0h 5m 0s with TBS (2/3).
Wash membrane for 0h 5m 0s with TBS (3/3).
Image membranes using a Licor Odyssey Infrared Imager.
