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 [http://www.pall.com/main/Laboratory/Product.page?id=20000 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 [http://www.pall.com/main/Laboratory/Product.page?id=20000 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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