Pierce high capacity streptavidin agarose

HEK 293T cells were co-transfected with FLAG-cGAS and FLAG-Cap or the empty expression vector using Lipofectamine LTX Reagent at 80% cell confluence and following the manufacturer's instructions. After 24 h, the transfected cells were transfected with 2 μg biotin-labeled DNA (ISD) (5′-biotin-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3′) for another 2 h, and the cells were washed three times with phosphate-buffered saline and lysed with RIPA buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, and 1% NP40) containing 5% glycerol and protease inhibitors (Complete Mini, Roche). The lysates were incubated with 20 μl Pierce™ High Capacity Streptavidin Agarose (Thermo Scientific) for 1 h at 4°C. The beads were recovered by centrifugation (3,000 × g, 1 min, 4°C), and the supernatant was discarded. Immunoprecipitation (IP) preparations were washed three times with lysis buffer, and the beads were recovered by centrifugation (3,000 × g, 1 min, 4°C). Proteins were eluted by boiling in 2 × SDS polyacrylamide gel electrophoresis (PAGE) loading buffer. The precipitated samples were analyzed by SDS-PAGE followed by immunoblotting.

A milliliter (mL) of streptavidin beads (Pierce High Capacity Streptavidin Agarose, Thermo Scientific Inc, USA) were equilibrated with 5 ml of BPRB for three times. The equilibrated beads were mixed with the 0.22 to 0.45 g of total cellular proteins and incubated at room temperature for at least 6 h and washed three times using Biotinylated Protein Washing Buffer 1 (BPWB1), including 8 M urea, 0.2 M NaCl, 0.5% SDS, 100 mM Tris (pH 7.4), three times with BPWB1 plus 2% SDS, and 8 to 10 times with one × PBS. In each step of washing, the streptavidin beads were collected by centrifugation at 1000g for 2 min. Biotinylproteins were eluted by an elution buffer containing 2% SDS, 3 mM biotin, and 15 mM Tris (pH 6.8) by rotating the solution at room temperature for 15 min, followed by another 15 min of heating at 96 °C. After 1 min of centrifugation at 1000g, the supernatant containing biotinylproteins was collected and vacuum-dried by Speed Vac (SPD1030, Thermo Scientific Inc) to be concentrated, which was consequently supplemented with 30% glycerol and 1% β-mercaptoethanol, leading to a final concentration of biotinylproteins to one to 2 μg/μl.