Team:Bielefeld-Germany/Protocols/Immobilization

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Immobilization

Contents

Immobilization

Immobilization on silica beads

  • Suspend silicium dioxide beads in recrystallization buffer (0.5 mM Tris-HCl, pH 9, 10 mM CaCl2) and mix it with the 1 mg/mL lacasse solution
    • Ratio of beads to protein should be 1 to 1000
    • 0.1 mg/mL final protein concentration
    • Contact with recrystallization buffer will start assembly of SbpA
  • Incubate on vertical rotator at room temperature for 4 h
  • After incubation: centrifuge down the beads (1 min, > 15,000 g), wash them twice with ddH2O and store them afterwards in ddH2O at 4 °C in the dark

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.

Activity measurements

Activity measurement of beads

Activity measurement of supernatant

Activity measurements of the supernatants see [here]



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