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Bio-Panning

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  1. Coat the wells of a 96-well ELISA plate with 2 μg of target antigen in 50 μL of 0.1M NaHCO3, pH8.6, and incubate overnight at 4 . Shake out the coating solution and block the wells by adding 300 μL blocking buffer. Seal and incubate overnight at 4 .
  2. Shake out the blocking buffer and add 50 μL of freshly prepared phage library to each well (4 wells). Seal the plate and incubate for 2 hr at 37 .
  3. Shake out the phage solution and wash 5 times by ELISA plate washer machine (in the second round wash 10 times, and in the third, fourth, fifth rounds wash 10-15 times).
  4. After shaking out the final washing solution, add 50 μL of 0.1M glycine-HCl, pH 2.2, incubate for 10 min at 37 . Pipet 10 times vigorously up and down and transfer the eluate to a microfuge tube containing 6 μL of neutralizing solution (1M Tris). Transfer the eluates to the 2-mL XLI-Blue cells and incubate at 37  for 30 min.
  5. Add 6 mL of pre-warmed SB medium and 1.6 μL of 100 mg/mL ampR. Shake the 8-mL culture at 250 rpm for 1hr at 37 , add 2.4 μL of ampR, and shake for an additional hr at 250 rpm and 37 .
  6. Add 2 mL of M13KO7 helper phage to the 8-mL culture and transfer to a flask. Add 90 mL of SB medium and 46 μL ampR. Shake the 100-mL culture at 250 rpm for 1.5 hr at 37 .
  7. Add 100 μL of 50 mg/mL kanR and continue shaking overnight at 250 rpm at 37 .
  8. Spin at 3,000 g for 15 min at 4 . Add 25 mL 5*PEG/NaCl, and store on ice for 30 min.
  9. Spin at 15,000 g for 15 min at 4 . Discard the supernatant, drain the bottle by inverting on a paper towel, and wipe off the remaining liquid from the upper part of the centrifuge bottle with a paper towel.
  10. Re-suspend the phage pellet in 2 mL of 1% BSA in TBS by pipetting up and down along the side of the centrifuge bottle. Transfer the suspension to a 1.5 mL tube. Vortex and spin at full speed in a micro-centrifuge for 5 min at 4 . Keep the supernatant at 4  for next round panning.
  11. 4-5 rounds of panning should be run.

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