α flag

HeLa cells were transfected with 1 μg DNA of each of the FLAG*−3A and an empty control plasmid. At 24 h post-transfection, cells were lysed using mild sonication in IP lysis buffer (20 mM HEPES, 1% (w/v) Triton X-100, 2 mM Magnesium chloride, 25 mM Sodium chloride, 110 mM Potassium acetate, and 0.2% (v/v) antifoam B), and clarified by centrifugation at 100,000 g for 3 min. Total protein concentrations were measured using BCA assay and 100 μg of lysate from each sample was used in the immunoprecipitation. 8 μg of α-FLAG (F3165, Sigma Aldrich) was conjugated to 10 mg epoxy-coated M-270 magnetic beads (ThermoFisher Scientific) (Cristea and Chait, 2011 (link)). The α-FLAG conjugated beads were washed and re-suspended in IP lysis buffer, and 3 mg bead aliquots were added to the clarified lysates. Lysates were incubated with magnetic beads overnight at 4 °C. After three washes with IP lysis buffer, bound proteins were eluted with 50 μl 1× LDS sample buffer and resolved on 4–20% Bis-Tris NuPAGE gel. Additionally, ~10% of the eluate volume was resolved on a 3–8% Tris-Tricine gel to confirm expression and enrichment of the immunoprecipitated 3A-FLAG* bait. Proteins were transferred to PVDF membranes and immunoblotted with α-GBF1 (ab86071; abcam, at 1:1000 dilution) and HRP conjugated α-FLAG (A8592; Millipore Sigma 1:2000 dilution). The experiment was performed in triplicate.