I've been trying to conjugate anti-biotin to SAMs of 3-mercaptopropanoic acid (MPA) (deposited on silver films), by first activating the COOH group using NHS (n-hydroxy succinimide) and EDC (ethyl dimethylaminopropyl carbodiimide) in buffer solution, and then rinsing in more buffer and drop coating the slides with the antibody solution (I would like to immerse the slides, but the antibody is too expensive!) for 90 mins at 4 degrees, then rinsing with buffer. I then do spectroscopy on the slides to see if the antibodies have conjugated - I'm not getting great results though!
I've been using buffer at pH 5.5 throughout (with KH2PO4, K2HPO4 and NaCl).
Am I doing anything obviously wrong/ do you have any tips/advice? Anything you can tell me will help, since I'm a physicist by trade and all this biochemistry is very new to me!