Team:Bielefeld-Germany/Protocols/Immobilization

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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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Fifteen students from different fields of study from the Bielefeld University aspired to complete a project called TOXIC COMPOUNDS IN NATURAL WATER - A CASE FOR LACCASE. In his Wiki we document our achievements and our approach.

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 *CPC-(controlled pore carrier) Silica beads, aminopropyl derivate (2-aminopropyl-triethosysilane), pore size: 500 A, 30-45 mesh
-Laccase was immobilized on the CPC-silica beads
+Immobilization of 1mL protein solution on 0,12g CPC-silica beads
-,12 g of beads were immersed in 1 mL 2.5% glutaraldehyde and put under light vacuum for 2 hours in order to degas the beads and allow as much bead surface area as possible to be coated with aldehyde groups.
+*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.
-The beads were washed (2 times with 1 ml) with Britton-Robinson-buffer (pH 5) and then immersed in a stock laccase solution (i.e., 1 mg of laccase in 1 mL of buffer prepared the same day) for at least 36 hours at 4° C.
-After washing the beads with buffer again (2 times with 1 mL), the beads were rinsed with 1 mL 0.5M sodium chloride in order to prevent sorption of the enzyme onto the beads, since only the measurement of reactions catalyzed by covalently bonded enzyme was desired.
-The beads were again washed with buffer and then immersed in 1 mL of 2.5 mg/mL glycine for at least 18 hours at 4° C.
-They were than washed, first with buffer, then 0.5 M sodium chloride then buffer, and finally stored in buffer at 4° C until use.
 All reagents used in the immobilization process were made in Britton-Robinson-buffer.