NK cells were treated with SNAP as described above. 20 μg of anti-nitrotyrosine beads (Millipore 16310) were added to 50 μg of protein lysate and incubated overnight at 4°C. The beads were washed with cold PBS and re-suspended in 2X Laemelli buffer and boiled. The sample was loaded on a SDS gel for protein expression by immunoblot analysis. The membrane was probed with an anti-Erk antibody (Cell Signaling) (28 (link)).
Protocol detail
Detecting Nitrotyrosine Protein Modifications
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Anti antibody
Buffer
Cold
Immunoblot analysis
Membrane
Nitrotyrosine
Nk cells
Protein
Corresponding Organization : The Ohio State University
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Variable analysis
independent variables
- SNAP treatment
- Nitrotyrosine-modified proteins
- Erk protein expression
- Protein lysate concentration (50 μg)
- Incubation time (overnight)
- Incubation temperature (4°C)
- Washing buffer (cold PBS)
- Gel electrophoresis (SDS-PAGE)
- Immunoblot analysis
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