Activated beads usually in a lyophilized formed.
Qucikly wash the bead with 10 vol. of cold 0.1N HCl then 10 vol of
distil water. Suction dry the bead to a wet cake and mixt with equal
vol. of avidin solution (in Na2CO3, pH9.5 to 10), constant shaking for
30 min to 60 min. Block the excess of the active isourea with
ethanolamine for another 60 min. Wash the bead with PBS then store at
4oC with preservative (NaN3).
Note: constant leaching of the avidin is common when cyanogen bromide is
the activated agen, but it is highly efficient.
Recommended the other activated matrix:
activated ECH, which is first attached a caroxilic acid to the agarose
via a secondary amine (react with epoxide) then the free carboxyl group
was actvated with N-hydroxyl succinimide and form an active ester. The
abundant primary amine of avidin will form high stable amide linkage
with the beads. Beside the secondary amine linkage is resistant of
leaching.
In article <Pine.SOL.4.05.9904081221200.17950-100000 at granby>,
rczci at unix.ccc.nottingham.ac.uk says...
>>Does anyone have a method for coating CNBr-sepharose with avidin?
Thanks.
>>>>>
