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Revision as of 00:48, 25 September 2012 (view source) NadineL (Talk | contribs) ← Older edit		Revision as of 00:51, 25 September 2012 (view source) NadineL (Talk | contribs) Newer edit →
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	{{Team:Bielefeld/Head}}		{{Team:Bielefeld/Head}}
-	== 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	+	== 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
		+
-	*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 ==		== Britton-Robinson-buffer ==
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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
		+
		+
		+
		+
		+
		+
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-	== Recrystallization buffer SbpA ==
-	*0.5 mM Tris-HCl, pH 9.0
-	*10 mM CaCl2
-	== Hanks Buffered Saline Solution (HBSS) ==	+	== 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.
		+
-	*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
	{{Team:Bielefeld/Sponsoren}}		{{Team:Bielefeld/Sponsoren}}

---

Revision as of 00:51, 25 September 2012

Contents 1 Recrystallization buffer SbpA 2 Hanks Buffered Saline Solution (HBSS) 3 Britton-Robinson-buffer 4 Immobilization on silica beads 5 Immobilization with CPC silica beads

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 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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