Team:Bielefeld-Germany/Protocols/Immobilization
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*CPC-(controlled pore carrier) Silica beads, aminopropyl derivate (2-aminopropyl-triethosysilane), pore size: 500 A, 30-45 mesh | *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 | |
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+ | *Immersing beads in 1 mL 2.5% glutaraldehyde under light vacuum for 2 hours. | ||
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+ | *Washing beads (2 times with 1 ml) with Britton-Robinson-buffer (pH 5). | ||
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+ | *Immersing 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. | ||
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+ | *Washing the beads with buffer again (2 times with 1 mL). | ||
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+ | *Adding 1 mL 0.5M sodium chloride. | ||
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+ | *washing with buffer again | ||
+ | *Immersing in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C. | ||
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+ | *Washing first with buffer, then winth 0.5 M sodium chloride then buffer again, and finally stored in buffer at 4° C until use. | ||
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All reagents used in the immobilization process were made in Britton-Robinson-buffer. | All reagents used in the immobilization process were made in Britton-Robinson-buffer. | ||
Revision as of 00:26, 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
- Immersing beads in 1 mL 2.5% glutaraldehyde under light vacuum for 2 hours.
- Washing beads (2 times with 1 ml) with Britton-Robinson-buffer (pH 5).
- Immersing 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.
- Washing the beads with buffer again (2 times with 1 mL).
- Adding 1 mL 0.5M sodium chloride.
- washing with buffer again
- Immersing 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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