HA immunoprecipitation was done as previously described49 (link). Cells were washed once with ice-cold PBS and harvested in immunoprecipitation buffer (20 mM HEPES pH 7.9, 200 mM NaCl, 0.5 mM EDTA, 10% glycerol, 0.2% NP-40) supplemented with phosphatase and protease inhibitors (Sigma), lysed for 30 min at 4 °C and cleared by centrifugation. For immunoprecipitation, HA-coupled magnetic beads (Pierce Thermo Fisher Scientific) were washed with IP buffer, added to the lysate and incubated for 3 h at 4 °C. Elution was performed in 40 µl 1x LDS buffer (NuPAGE Thermo Fisher Scientific) by incubating at 37 °C with shaking for 30 min.
Protocol detail
HA-Immunoprecipitation Protocol for Protein Isolation
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Buffer
Cells
Centrifugation
Cold
Edta
Glycerol
Hepes
Immunoprecipitation
Nacl
Np 40
Phosphatase
Protease inhibitors
Corresponding Organization :
Other organizations : University of Würzburg, Goethe University Frankfurt, German Cancer Research Center, Heidelberg University, Technical University of Munich
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Variable analysis
independent variables
- None explicitly mentioned
- Protein-protein interactions (immunoprecipitation of HA-tagged proteins)
- Cell lysis buffer composition (20 mM HEPES pH 7.9, 200 mM NaCl, 0.5 mM EDTA, 10% glycerol, 0.2% NP-40, with phosphatase and protease inhibitors)
- Incubation time and temperature for immunoprecipitation (3 h at 4 °C)
- Elution method (incubation in 1x LDS buffer at 37 °C with shaking for 30 min)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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