Protein co-IP assays were performed following the previously published protocol (62 (link)). Briefly, protein A/G magnetic beads (Cat. # 88802, Thermo Scientific) and anti-FLAG M2 magnetic beads (Cat. # M8823, Sigma-Aldrich) were washed with 1× RIPA buffer containing protease inhibitor cocktail. Cellular lysates were precleared with the empty magnetic beads for 1 h at 4°C on a 360° tube rocker. The cell lysate was incubated with anti-FLAG M2 magnetic beads for pull-down of FLAG-Nsp14 protein at 4°C overnight with constant rotation. Protein immunocomplexes were washed by RIPA buffer and boiled in SDS loading buffer containing 5% 2-mercaptoethanol, followed by protein immunoblotting. A normal mouse IgG antibody (Cat. # sc-2025, Santa Cruz) was used as the control in parallel.
Protocol detail
Protein Co-Immunoprecipitation Assay
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Assays
Buffer
Cell
Flag
Igg antibody
Mercaptoethanol
Mouse
Protease inhibitor 1
Protein
Protein a
Protein g
Ripa
Corresponding Organization : The Ohio State University Wexner Medical Center
Other organizations : Columbia University Irving Medical Center, University of Alabama at Birmingham
Top 5 similar protocols
Variable analysis
independent variables
- Pull-down of FLAG-Nsp14 protein using anti-FLAG M2 magnetic beads
- Protein immunocomplexes
- Protein A/G magnetic beads
- RIPA buffer containing protease inhibitor cocktail
- Preclearing of cellular lysates with empty magnetic beads
- Incubation time and temperature (4°C overnight with constant rotation)
- Washing of protein immunocomplexes with RIPA buffer
- Boiling of protein immunocomplexes in SDS loading buffer containing 2-mercaptoethanol
- Normal mouse IgG antibody as a control
- Pull-down of FLAG-Nsp14 protein using anti-FLAG M2 magnetic beads
- Normal mouse IgG antibody
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