Hi Jahan, Maybe you are using a new model of BiaCore. Our old BiaCore X never cares to tell us how to do our experiment. However if your RU did go below that of the empty chip, then maybe you really didn't get anything conjugated.
The pH 3.8 condition is not good for the amine coupling reaction. The reaction will be very inefficient when pH drops below 5. You can try conjugating at higher pH, such as 6, 5.5 or 5. If you can not get protein adsorbed to the surface by electrostatic attraction, then simply increase the protein concentration - 0.05, 0.1mg/mL, or as much as you can afford to use, and extend the time of reaction (you can slow down or even stop the flow of protein to get some "incubation"). You should see at least 100 or 200 RU increase after a successful coupling for your 15kD protein. How many Lys do you have in the 15kDa protein? If you have very few Lys, and the protein is made in E coli, you can add a short poly Lys tail to either ends of the protein. This also has an additional advantage if some of the Lys residues are located in the binding surface. Zhijie From: Jahan Alikhajeh Sent: Wednesday, May 08, 2013 3:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic (SPR) Dear pals, Sorry for my off-topic question but its been a while I am challenging to get a 15 kDa protein coated on CM5 chip. Calculated PI of protein is 5.9 & I tried Acetate buffer pH5.2 and 3.8 for coating under normal procedure of Biacore. After immobilization I saw this message: response level decreased! This happened for me several times. Does anyone have any idea what's going on? Cheers, Jahan
