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Coating of CNBr-activated resins sepharose 4B with Antigen

  1. Before starting the coupling, the antigen was dialysed against coupling buffer at least one hour (coupling buffer: 0.1 M NaHCO3 pH 8.3, 0.5 M NaCl).
  2. Amount of resin to use: 5 - 10 mg protein / ml resin (but for small protein like ED-B or domains of Tenascin 2 mg protein / ml resin).
  3. To calculate the amount of pulver to use: 1 g resin = 3.5 ml gel (ex.: 35 mg ED-B = 17.5 ml gel = 5 g resin).
  4. 2 mg of resin were weighed (for 12 g ED-B).
  5. The resin was resuspended in some ml 1 mM HCl.
  6. This gel was washed in a Gutch filter with 1 mM HCl (200 ml / g resin).
  7. The washed resin was centrifuged at 2500 rpm for 3 minutes and supernatant was removed.
  8. The resin was resuspended in coupling buffer.
  9. Now the resin is ready to be coupled with the antigen.
  10. The dialysed protein was mix together with the resin in one or more Falcon tubes (ex.: 15 ml protein + 25 ml coupling buffer containing resin).
  11. The mix can rotate one hour at RT or over night at 4°C (do not use magnatic stirrers)
  12. To control the capacity of the resin, measure the optical density at 280 nm
  13. Excess ligand was washed away in a Gutch filter using at least 5 gel volumes of coupling buffer
  14. Any remaining active groups should be block by leaving the resin for 2 hours at RT in 0.1 M Tris - HCl pH 8 (without moving)
  15. The resin was now centrifuged at 2500 rpm for 2 minutes and supernatant was removed.
  16. Once more the resin was washed in a Gutch filter with pH change: 0.1 M NaAcetate, 0.5 M NaCl, pH 4 (with Acetic acid) (ca. 50 ml), and 0.1 M Tris, 0.5 NaCl, pH 8 (with HCl) (ca. 50 ml), for three times.
  17. The resin was then resuspended in PBS, 1000x Pefablock, 0.1% Saodium Azide, and stored at 4°C

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