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Team:Bielefeld-Germany/Protocols/Immobilization
From 2012.igem.org
(Difference between revisions)
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| - | + | = Buffers = | |
| - | = Recrystallization buffer SbpA = | + | == Recrystallization buffer SbpA == |
*0.5 mM Tris-HCl, pH 9.0 | *0.5 mM Tris-HCl, pH 9.0 | ||
*10 mM CaCl2 | *10 mM CaCl2 | ||
| - | = Hanks Buffered Saline Solution (HBSS) = | + | == Hanks Buffered Saline Solution (HBSS) == |
*0.137 M NaCl | *0.137 M NaCl | ||
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*4.2 mM NaHCO3 | *4.2 mM NaHCO3 | ||
| - | = Britton-Robinson-buffer = | + | == Britton-Robinson-buffer == |
*0.1 M acetic acid | *0.1 M acetic acid | ||
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adjusted to the desired pH by addition of 1 N sodium hydroxide | adjusted to the desired pH by addition of 1 N sodium hydroxide | ||
| - | + | = Immobilization = | |
| - | = Immobilization on silica beads = | + | == 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 | *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 | ||
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*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 | *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= | + | == Immobilization with CPC silica beads== |
*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 | ||
Revision as of 00:55, 25 September 2012
Contents |
Buffers
Recrystallization buffer SbpA
- 0.5 mM Tris-HCl, pH 9.0
- 10 mM CaCl2
Hanks Buffered Saline Solution (HBSS)
- 0.137 M NaCl
- 5.4 mM KCl
- 0.25 mM Na2HPO4
- 0.44 mM KH2PO4
- 1.3 mM CaCl2
- 1.0 mM MgSO4
- 4.2 mM NaHCO3
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
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.
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