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Team:Bielefeld-Germany/Protocols/Immobilization
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*Immerse beads in 1 mL 2.5% glutaraldehyde under light vacuum for 2 hours. | *Immerse beads in 1 mL 2.5% glutaraldehyde under light vacuum for 2 hours. | ||
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*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). | ||
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*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. | *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. | ||
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*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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*Add 1 mL 0.5M sodium chloride. | *Add 1 mL 0.5M sodium chloride. | ||
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*wash with buffer again | *wash with buffer again | ||
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*Immerse in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C. | *Immerse in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C. | ||
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*Wash first with buffer, then winth 0.5 M sodium chloride then buffer again, and finally stored in buffer at 4° C until use. | *Wash 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 00:41, 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.
- 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.
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
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-1 S-layer solution
- ratio of beads to protein can be varied
- 0.1 mg mL-1 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
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