We performed co-immunoprecipitation using Pierce Anti-HA Magnetic Beads (Life Technologies) or using Dynabeads-protein G (Thermo) magnetic beads with primary antibodies (anti-GFP Genetex No#GTX113717; anti-Sec5 Proteintech No#12751-1-AP) cross-linked using dimethyl pimelimidate (Life Technologies).
For the production of whole-cell lysates, cells were lysed at 4 °C in lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl, 0.25% sodium deoxycholate [v/v], protease inhibitor cocktail [1× EDTA-free protease inhibitors, Sigma], 10 mM NaF). 1/10 part of the clarified lysate was saved as an input fraction, and the rest was subjected to immunoprecipitation.
After adding beads, binding of the protein of interest was performed overnight with gentle rotation at 4 °C. The next day, beads were washed 4 × 10 min at 4 °C in the same lysis buffer to remove unbound proteins, and complexes were eluted off the beads using 2x SDS sample buffer at 37C for 30min. We analyzed the composition of eluent using the SDS-PAGE and Western Blot method.
For the production of whole-cell lysates, cells were lysed at 4 °C in lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl, 0.25% sodium deoxycholate [v/v], protease inhibitor cocktail [1× EDTA-free protease inhibitors, Sigma], 10 mM NaF). 1/10 part of the clarified lysate was saved as an input fraction, and the rest was subjected to immunoprecipitation.
After adding beads, binding of the protein of interest was performed overnight with gentle rotation at 4 °C. The next day, beads were washed 4 × 10 min at 4 °C in the same lysis buffer to remove unbound proteins, and complexes were eluted off the beads using 2x SDS sample buffer at 37C for 30min. We analyzed the composition of eluent using the SDS-PAGE and Western Blot method.
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