I am running some experiments and I experience non-specific interaction with my reference flow cells, that are blocked with ethanolamine from the Biacore Immobilization kit. I would like to try a different blocking approach, by blocking both the active and reference flow cells with mPEG-NH2 of 350 Da.
I was wondering I anyone of you has experience with this and can give me some advice in what buffer and at which pH immobilization of mPEG-NH2 is the most efficient.
Many thanks in advance!
Immobilization with mPEG-NH2 in stead of EtOH-NH2
07 Mar 2014 23:00 #2
I tested blocking with mPEG-NH2 by dissolving a high concentration of mPEG-NH2 in different buffers, Na-acetate pH 5,5; HBS pH 7.4 and carbonate buffer pH 9. 600s injection of mPEG-NH2, diluted 1:10 directly out of stock in HBS pH 7.4 , resulted in blocking of both reference and active flow cells.
Since I used a low molecular weight PEG (350Da) I did not see a significant RU gain after deactivation (in analogy with ethanolamine). I tested this blocking by injecting high antibody concentrations on the activated flow cell after the blocking with mPEG-NH2, and I did not have any covalent blocking of the mABs, which proves the activated surface was deactivated sufficiently.
This allowed me to perform kinetic analysis with mABs in solution and immobilized antigen, whereas the ethanolamine blocking did not make this kind of set-up possible, due to high non-specific binding of different mABs to the ethanolamine reference.