Hi, I wonder what is the proper immobilization level for my experiments in Biacore. I was using about 1000 RU and it was ok for kinetics measurements. However, a different protein was immobilized at about 1000 RU also and it did not work at all, I mean protein was immobilizzed but no binding was observed... Any suggestion??
You should always take the molecular mases of your molecules into the account. Use formula Rmax= MWanalyte/MWligand*Rimm*S, where S is stoichiometric ratio and Rimm is immobilization level. Adjust Rimm to obtain Rmax (maximum binding of analyte) values between 50-100 RU (for kinetic analysis).