Team:Bielefeld-Germany/Protocols/Immobilization
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(→Immobilization with CPC silica beads) |
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Immobilization of 1mL protein solution on 0,12g CPC-silica beads | 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). | *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). | *Wash the beads with buffer again (2 times with 1 mL). | ||
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*wash with buffer again | *wash with buffer again | ||
- | * | + | |
+ | *Immerse in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C. | ||
*Washing first with buffer, then winth 0.5 M sodium chloride then buffer again, and finally stored in buffer at 4° C until use. | *Washing first with buffer, then winth 0.5 M sodium chloride then buffer again, and finally stored in buffer at 4° C until use. |
Revision as of 00:28, 25 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
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.
- Washing 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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