10 kd ultrafiltration tube

HEK293T cells were stably transduced with packaged lentivirus to express RGD-C1C2 or a scrambled ACDCRDGCFC peptide fused to C1C2 (Scr-C1C2). Cells were cultured for 48 h, and the conditional medium was harvested. After depletion of cells and debris by centrifugation at 10,000 g for 20 min at 4 °C, the supernatant was concentrated using a 10-kD ultrafiltration tube (Millipore). The concentrate was incubated with 0.5% Triton X-100 for 30 min to disrupt protein-EV interactions. RGD-C1C2 or Scr-C1C2 was purified using a His-tag Protein Purification Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions and quantified by BCA protein assay (Pierce). Western blot analysis was performed using an anti-HA antibody (Sigma, St. Louis, USA) to detect recombinant RGD-C1C2. For EV decoration, 0.5 mg/mL EVs were incubated with 1 μg/mL RGD-C1C2 or Scr-C1C2 for 15 min at room temperature (RT). Unbound proteins were removed by ultracentrifugation at 200,000 g for 90 min at 4 °C.

320 μl of high-abundance protein-depleted sample was added to a 10 kD ultrafiltration tube (Millipore), 12,000g, centrifuged for 10 min; added 200 μl of 8 M Urea, 12,000g, centrifuged for 10 min, repeated once, and finally 50 μl of 8 M urea were added. Then the sample was added into a 96-well plate. 10 mM DTT solution was added and reacted at 37 °C for 30 min; IAM solution was added to a final concentration of 20 mM, and reacted at 37 °C for 30 min at room temperature in the dark for 30 min; DTT solution was added to a final concentration of 10 mM to quench the reaction. 1 μg of LysC was added to each sample, incubate at 37 °C for 2 h; 1 μg Trypsin was then added and incubated at 37 °C overnight; finally 10 μl of 10% TFA was added to terminate the reaction.

We placed 2 μL of phage culture supernatant of the positive clones in logarithmic growth into 400 μL of HB2151 culture solution phase and shook the solution at 150 rpm for 30 min at 37 °C. Then, the bacteria were inoculated onto LB-A plates and incubated at 30 °C overnight with TG1, HB2151 and recombinant phages used as controls. Next, a single colony was inoculated into 2 × YT-AG culture medium and shaken overnight at 250 rpm at 30 °C to OD600 nm ≈ 0.5–0.8. We then added 1.0 mmol/L IPTG to induce protein expression with overnight shaking at 250 rpm and incubation at 30 °C. The bacterial pellet was collected by centrifuging at 8000 rpm for 10 min at 4 °C, and the pellet was used to obtain antibodies in the periplasm with a final concentration of 0.5 mU of polymyxin B schizothrix. The bacterial periplasmic extracts were filtered with 0.45 μm membranes and stored at 4 °C or used for detection and purification.
The purification was performed according to the instructions of the HisPur Ni NTA kit (Smart-Lifesciences, Changzhou, China). After purification, the collected liquid was concentrated via ultrafiltration using a Millipore 10 KD ultrafiltration tube with centrifugation at 5000 rpm for 15 min at 4 °C.