Team:Bielefeld-Germany/Protocols/Immobilization

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Immobilization with CPC silica beads

  • CPC-(controlled pore carrier) Silica beads, aminopropyl derivate (2-aminopropyl-triethosysilane), pore size: 500 A, 30-45 mesh

Immobilization of 1mL protein solution on 0,12g CPC-silica beads

  • Immerse beads in 1 mL 2.5% glutaraldehyde under light vacuum for 2 hours.
  • Wash beads (2 times with 1 ml) with Britton-Robinson-buffer (pH 5).
  • Immerse in stock laccase solution (i.e., 1 mg of laccase in 1 mL of buffer prepared the same day) for at least 24 hours at 4° C.
  • Wash the beads with buffer again (2 times with 1 mL).
  • Add 1 mL 0.5M sodium chloride.
  • wash with buffer again
  • Immerse in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C.
  • Wash first with buffer, then winth 0.5 M sodium chloride then buffer again, and finally stored in buffer at 4° C until use.

All reagents used in the immobilization process were made in Britton-Robinson-buffer.

Britton-Robinson-buffer

  • 0.1 M acetic acid
  • 0.1 M boric acid
  • 0.1 M phosphoric acid

adjusted to the desired pH by addition of 1 N sodium hydroxide


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