Example – obtaining antibody:30 kDa protein 1:1 conjugates. We suggest modifying the 30 kDa protein with HyNic and the antibody with 4FB. For HyNic, use 15–20X mole equivalents to have 2–3 per protein. For 4FB, use 10–20X mole equivalents to have 3–5 per antibody. Make a 1 mg/mL solution of each for modification. After a 2 hr modification, pass 130 µL, corresponding to 130 µg over the desalting column – should get about 100 µg of modified protein. With these 2 tubes, make some combinations for your conjugates (incubating 2 hr with 10X TurboLink buffer (Cat. No. S-2006-105)) and run the products on a gel to see which ratios give the best 1:1 conjugate. Scale up your conjugations after getting results from the gel.
Pair-wise combinations – can set this up, using both modification conditions for both your antibody and protein:
0.5 / 1
1 / 1
1 / 0.5
For quenching the conjugation reaction, add a 50-fold molar excess of sulfobenzaldehyde to the reaction to react with any excess HyNic groups.