Team:Bielefeld-Germany/Protocols/Immobilization

From 2012.igem.org

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 measurements of laccases bound on beads were performed using AcroPrepTM 96-well Filter Plates.

  • 158 µL deionized H2 and 40 µL 100 mM sodium acetate were added to the samples containing dry beads.
  • The whole sample volume was transfered into the AcroPrepTM 96-well Filter Plates.
  • The reaction was induced adding 0.1 mM ABTS at room temperature.
  • The reaction was stopped by centrifugation at 13000 g for 30s.
  • Oxidized ABTS was detected photometrically at 420 nm.

Alternative measurement of beads

Activity measurements of laccases bound on beads were alternatively performed while using the photometer Thermo biomate3 UV-Vis Spectrophotometer and classic plastic cuvettes.

  • Beads were measured under substrate saturation in a total volume of 500 µl.
  • The reaction was induced adding the specific ABTS concentration at room temperature.
  • Mixing was realized through vortexing between measurements.
  • Substrate reduction was measured every 20 seconds for at least one hour.
  • Oxidized ABTS was detected photometrically at 420 nm.

Activity measurement of supernatant

Activity measurements of the supernatants see [here]



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