I want to conjugate protein A (43 kDa) to antibody IgG (150 kDa). We have both cross linkers (C6-SANH (HyNic) linker and C6-S-4FB linker). My protein is very sensitive to pH, buffer composition, temperature, and solvent. It’s very risky to incubate at room temperature for a long time. How many fold excess of linker would be suitable for this protein? Is it ok to incubate this reaction at 4 degrees (C) for 2 hr for modification? Please suggest the antibody IgG (150 kDa) amount. I will use C6-S-4FB crosslinker for the modification of antibody. For the conjugation reaction, which molar ratio would be suitable for best conjugation of both modified proteins (I am not sure it depends upon the amount of modified protein or crosslinker attached in modifying protein). The molecular weight of my protein A (43 kDa) is approx three times lesser than the antibody IgG (150 kDa). Is it ok to incubate the conjugation reaction for 12 hr at 4 degrees (C) with pH near 7 for best conjugation?

All of our modification data is at room temperature. While modification at 4 degrees may work, we cannot guarantee success. For the modification, we would suggest 15 equiv at 2 mg/mL protein A concentration. We suggest 10 equiv at 2 mg/mL. For the conjugations, we recommend a 1/1 mol/mol ratio. However, we suggest trying 1/1.5 and 1/2 antibody/protein A ratios on an initial small scale (~10 µg) reactions that can be analyzed by PAGE. Use 10X TurboLink buffer in the solution to optimize the conjugation reaction.