Coupling reactions are typically performed at room temperature overnight by
using N, N-dimethylformamide (DMF) and Tris-HCl or PBS Buffer (pH 7.0–7.5) as
solvent. It may be advisable to carry out the reduction of disulfide bonds by
adding a 10-fold molar excess of a reducing agent such as DTT or TCEP. If DTT is
used, then dialysis is required to remove the excess of DTT prior to the
addition of the reactive malemide.
Example Reaction: Coupling of BG-Maleimide to a 5´
thiol modified Oligonucleotide:
Figure 1. Reaction of
BG-Maleimide with thiol groups. The 5´-thiol modified oligonucleotide (43 nmol)
was reduced by incubation for 1 hour at room temperature with 10 mM DTT in 200
µl 20 mM Tris-HCl pH 8.5. The DTT was removed by gel filtration and the
oligonucleotide eluted in PBS buffer (pH 7.4). The most concentrated fractions
were combined giving a total of 800 µl. Three hundred microliters of
BG-maleimide solution (2.5 mM in DMF) was added and the reaction mixture
incubated at room temperature for 1 hour. The reaction mixture was diluted with
water to a total volume of 2 ml and excess maleimide removed by gel filtration.
The BG-maleimide-oligonucleotide conjugate was then purified by HPLC (solvent A:
0.1 M tetraethylammonium acetate pH 6.9 in water; solvent B:
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