Coating of CNBr-activated resins sepharose 4B with Antigen
- 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).
- 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).
- 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).
- 2 mg of resin were weighed (for 12 g ED-B).
- The resin was resuspended in some ml 1 mM HCl.
- This gel was washed in a Gutch filter with 1 mM HCl (200 ml / g resin).
- The washed resin was centrifuged at 2500 rpm for 3 minutes and supernatant was removed.
- The resin was resuspended in coupling buffer.
- Now the resin is ready to be coupled with the antigen.
- 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).
- The mix can rotate one hour at RT or over night at 4°C (do not use magnatic stirrers)
- To control the capacity of the resin, measure the optical density at 280 nm
- Excess ligand was washed away in a Gutch filter using at least 5 gel volumes of coupling buffer
- 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)
- The resin was now centrifuged at 2500 rpm for 2 minutes and supernatant was removed.
- 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.
- The resin was then resuspended in PBS, 1000x Pefablock, 0.1% Saodium Azide, and stored at 4°C
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