Anti flag antibody

Membrane lipid strips (Echelon Bioscience, #P-6002) were incubated with 5 mL Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) overnight on an orbital shaker at 4°C. The next day, the buffer was renewed, and the strip incubated for another hour. The strip was then incubated with a 5 mL solution of ~0.05 mg/mL IFT-A in buffer containing 20 mM HEPES pH 7.4, 5 mM MgSO₄, 1 mM CaCl₂, 50 mM KCl, and 1 mM DTT at room temperature with constant shaking. After 2 hrs, the protein solution was discarded, and the strip washed twice (5 min per wash) with a total of 10 mL blocking buffer (TBS with 0.001% tween-20 (TBST) and 5% milk). The strip was then incubated with 5 mL blocking buffer containing a 1:500 dilution of anti-FLAG antibody (BioLegend, #637301) overnight with constant shaking at 4°C. Next, the strip was washed three times with 10 mL TBST for a total of 30 min before incubating with a horseradish peroxidase-conjugated anti-rat secondary antibody (Cell Signaling Technology, #7077S) for 1 hr at room temperature. Finally, the strip was washed three times with TBST and once with TBS (10 mL per wash, each for 10 min). Signal was detected using the Novex ECL chemiluminescence kit (Thermo Fisher Scientific, #WP20005).