I have always used capture immobilization. It is the first time I'm going to do a Direct immobilization on a CM5 chip. Will use Amine coupling. My Ligand mol. wt. is ~28000. I want to have an RL of approx. 200 RU. Any suggestions on; 1) concentration of ligand to start? 30 ug/mL:? 2) Suggested contact time for the ligand; 3) Suggested time for NHS/EDC activation? 4) How long to block with Ethanolamine. Kindly comment. Thanks.