Team:Bielefeld-Germany/Protocols/Immobilization
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== Immobilization with CPC silica beads== | == Immobilization with CPC silica beads== | ||
Revision as of 20:10, 15 September 2012
Immobilization with CPC silica beads
- CPC-(controlled pore carrier) Silica beads, aminopropyl derivate (2-aminopropyl-triethosysilane), pore size: 500 A, 30-45 mesh
Laccase was immobilized on the CPC-silica beads 0,12 g of beads were immersed in 1 mL 2.5% glutaraldehyde and put under light vacuum for 2 hours in order to degas the beads and allow as much bead surface area as possible to be coated with aldehyde groups.
The beads were washed (2 times with 1 ml) with Britton-Robinson-buffer (pH 5) and then immersed in a stock laccase solution (i.e., 1 mg of laccase in 1 mL of buffer prepared the same day) for at least 36 hours at 4° C. After washing the beads with buffer again (2 times with 1 mL), the beads were rinsed with 1 mL 0.5M sodium chloride in order to prevent sorption of the enzyme onto the beads, since only the measurement of reactions catalyzed by covalently bonded enzyme was desired. The beads were again washed with buffer and then immersed in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C. They were than washed, first with buffer, then 0.5 M sodium chloride then buffer, 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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