I use the Biacore X (with two flow cells) and have some immobilization problems. I don`t get the same immobilization levels if I immobilize (by amine coupling) a 36 kDa protein on the one cell and a 26 kDa protein (control protein) on the other cell. I used the same concentration (in molar) for both proteins. How I can manage this? Is it possible injecting the ligand several times to get the wanted immobilization level after the NHS/EDC activation?
Thanks a lot!
Different immobilization levels are not a problem, but if you really want the same levels you can play with several parameters like: EDC/NHS activation time, protein concentration, protein buffer pH (choose 0.5 below the optimum to have a better control on the immob level), injection time... Remember that you can reactivate a surface w/ another EDC/NHS injection and immobilize more protein if you want.
And, of course, as you suggest you can immobilize with several consecutive injections, but rememeber that the activation of the surface doesnt last for ever and that reactivating is only effective a couple of times.
You have all this things in Maquart's book. I found it very useful!