Neutravidin agarose resin beads

HEK293 cells were plated on 6-well plates and transfected as described previously. Forty-eight hours later, cells were incubated for 15 min on ice, and all solutions and buffers used were prechilled. Each well was washed with PBS and then incubated for 1 h at 4 °C while rocking with 1 mg/ml EZ-Link NHS-SS-Biotin (Thermo Fisher Scientific) in PBS. Cells were then washed with PBS and incubated for 20 min at 4 °C with 100 mM glycine in PBS while rocking. After washing with PBS, cells were lysed using 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, pH 7.5 (lysis buffer), containing protease inhibitors (cOmplete protease inhibitor cocktail; Sigma-Aldrich). The lysates were centrifuged at 10,000g for 2 min, and the supernatant was incubated for 1 h at room temperature using end-over-end rotation with prewashed NeutrAvidin Agarose Resin beads (Thermo Fisher Scientific). The beads were washed with lysis buffer, captured proteins were eluted with 1× SDS sample buffer containing 5% β-mercaptoethanol and 1× protease inhibitors at 70 °C for 10 min and collected by centrifugation at 850g for 5 min.

The experiment procedure is based on our standard laboratory protocol as described [16 (link),63 (link)]. Briefly, transfected HEK293T cells were gently washed with room temperature PBS-Ca-Mg (PBS with 0.1 mM CaCl₂ and 1 mM MgCl₂) and then incubated with EZ-Link Sulfo-NHS-SS-biotin (Thermo Scientific 21331, Waltham, MA, USA) in PBS-Ca-Mg. The biotinylation reaction was then quenched with 0.1 M glycine in PBS-Ca-Mg. Cells were then collected and lysed in standard RIPA-PI buffer. Biotinylated proteins were purified by incubating the cell lysates overnight with high-capacity Neutravidin agarose resin beads (Thermo Scientific 29202). After incubation, the beads were washed with RIPA-PI to remove nonbiotinylated protein, and biotinylated protein was next eluted from the beads with Laemmli sample buffer containing β-mercaptoethanol. Samples were then subjected to standard immunoblot procedures.

Overnight cultures of C79-13 were subcultured in LB medium at 37°C for 4 h. Bacterial pellets were collected, washed using ddH₂O, and stored at −80°C. The pellets were resuspended in 20 ml lytic fluid buffer (20% of sucrose, 4% of CHAPS, and 40 mmol/l DTT) under 700 Mpa high-pressure crushing for 3 min. The bacteria were cracked by ultrasonication, and the cracking supernatant was collected after centrifugation. Biotinylated DNA and NeutrAvidin Agarose Resin beads (ThermoFisher, USA) were mixed and incubated at 25°C for 1 h. Following this, the probe-labeled beads were washed twice with TE and BS/THES buffers. The probe-labeled beads and supernatant lysates were then mixed and incubated with slow shaking at 25°C for 30 min. After washing four times with BS/THES buffer, the binding proteins were eluted from the beads with a NaCl concentration gradient. Finally, the beads were eluted with ddH₂O to release biotin-DNA for SDS-PAGE analysis. The experiments were performed three times and confirmed to be stable with similar bands by SDS-PAGE analysis. Finally, the proteins eluted with 500 nM and 750 nM NaCl buffer were submitted to GeneCreate (Wuhan, China) for mass spectrometry (MS).