小中大Protocol
1. Dissolve the protein to be activated in 0.05 M MES, 0.5 M NaCl, pH 6.0 (reaction buffer),
at a concentration of 1 mg/ml.
2. Add to the solution in step 1 a quantity of EDC and sulfo-NHS (Thermo Fisher) to obtain
a concentration of 2 mM EDC and 5 mM sulfo-NHS. To aid in aliquoting the correct
amount of these reagents, they may be quickly dissolved in the reaction buffer at a higher
concentration, and then a volume immediately pipetted into the protein solution to obtain
the proper molar quantities.
3. Mix and react for 15 minutes at room temperature.
4. Add 2-mercaptoethanol to the reaction solution to obtain a final concentration of 20 mM.
Mix and incubate for 10 minutes at room temperature. Note: If the protein being activated is sensitive to this level of 2-mercaptoethanol, instead of quenching the reaction
chemically, the activation may be terminated by desalting (step 5).
5. If the reaction was quenched by the addition of 2-mercaptoethanol, the activated protein may be added directly to a second protein or other amine-containing molecule for
conjugation. Alternatively, or if no 2-mercaptoethanol was added, the activated protein
may be purified from reaction by-products by gel filtration using a desalting resin. The
desalting operation should be done rapidly to minimize hydrolysis and recover as much
active ester functionality as possible. The use of centrifugal spin columns of some sort
may afford the greatest speed in purification (Thermo Fisher). After purification, add the
activated protein to the second molecule for conjugation. The second protein or other
amine-containing molecule should be dissolved in 0.1 M sodium phosphate, pH 7.5.
This will bring the pH of the coupling medium above pH 7.0 to initiate the active ester
reaction.
6. React for at least 2 hours at room temperature.
7. Remove excess reactants by gel filtration or dialysis.
