MEFs were seeded in 24-well plates at a density of ∼25 000 cells per well for 48 h and 50 000 cells per well for 24 h. Analyses were carried out as previously described (18 (link)). Cell lysates were separated at 80 V on 10% acrylamide gels for 2 h. Following transfer at 20 V for 1.5 h using Bolt Mini Blot Modules (Life Technologies) to Immobilon-FL (Millipore) membranes, membranes were blocked 30 min in Odyssey blocking buffer (LI-COR). The membranes were subsequently incubated overnight with 1:1000 mouse monoclonal anti-Viperin (MaP.VIP | MABF106, Millipore), rabbit anti-mouse p56 (gift from G. Sen, Cleveland Clinic, Cleveland, OH, USA), rabbit anti-mouse Mda5 (D74E4 | 5321, Cell Signaling Technology) or mouse monoclonal anti-β-tubulin (TU-06 | ab7792, Abcam). Finally, conjugated secondary antibodies with Alexa Fluor® 680 dye (Life Technologies) or IRdye800 (Rockland) were used to image the proteins at 700 or 800 nm with an Odyssey scanner (LI-COR).
Protocol detail
Western Blot Analysis of Immune Proteins
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Bolt Mini Blot Modules
Immobilon-FL
Odyssey blocking buffer
Alexa Fluor® 680 dye
IRdye800
Odyssey scanner
Acrylamide
Antibodies
Buffer
Cell
Gels
Immobilon
Irdye800
Mda5
Membranes
Mouse
Odyssey
Proteins
Rabbit
Tubulin
Corresponding Organization : Hudson Institute of Medical Research
Other organizations : Monash Medical Centre, University of Melbourne, Walter and Eliza Hall Institute of Medical Research
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Variable analysis
independent variables
- Cell seeding density: ~25,000 cells per well for 48 h and 50,000 cells per well for 24 h
- Protein expression of Viperin, p56, and Mda5 measured by Western blot analysis
- Cell culture duration: 24 h and 48 h
- Gel electrophoresis: 10% acrylamide gels, 80 V for 2 h
- Protein transfer: 20 V for 1.5 h using Bolt Mini Blot Modules
- Blocking: 30 min in Odyssey blocking buffer
- Primary antibodies: mouse monoclonal anti-Viperin, rabbit anti-mouse p56, rabbit anti-mouse Mda5, mouse monoclonal anti-β-tubulin
- Secondary antibodies: Alexa Fluor® 680 dye or IRdye800
- Positive control: Not specified
- Negative control: Not specified
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