Home Protocols Reaction Conditions for Chemical Coupling (S9153)

Reaction Conditions for Chemical Coupling (S9153)

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:

Coupling of BG-Maleimide

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: acetonitrile).