Protein A amine coupling to sensor chips

Hi fellow SPR enthusiasts,

Has anyone had experience with amine coupling Protein A to C1 chip (or CM5 or any other). I have been trying to immobilize ProA on my C1 chip and always ended up with Response bound about 8-20RU. I have tried acetate buffers with different pH (4;4.5;5; with pH 4 giving the best response) using the pH immobilization scout. Also tried two different concentrations of ProA (15ug/ml; 20ug/ml). I used the immobilization wizard method (7 min activation with EDC/NHS, 7 min ProA, 7 min ethanolamine quench; 10ul/min). C

Continued (accidentally posted without finishing)

I was thinking of trying to increase the immobilization time (from 7min to like 60min because I read someone did it for ProG). And also increase the concentration to like 50ug/ml. Hopefully that will make it immobilize more. Another variable that came into mind was changing the immobilization buffer (used acetate buffer). 

Any help would be greatly appreciated!

SPRofessional

Hi,
The C1 sensor chip is a flat surface without a dextran matrix to facilitate pre-concentration. The absence of a surface matrix will result in an immobilization yield that is approximately 10% of that obtained on Sensor chip CM5 under comparable conditions.
before the use of the C1 you can clean the surface with two injections of 1-2 minutes with freshly prepared 0.1 M Glycine-NaOH pH 12 with 0.3 % Triton X-100. Wash the system and proceed with immobilization.
Longer immobilization times can give you more protein A on the surface but don't expect too much on a C1 chip.
Since you metion the C1 chip I would recommend the Protein A chip which is ready to use and has probably a better performance sbut does not bind Fab fragments.

Kind regards
Arnoud
 

Thank you so much for your answer. I did do the C1 preconditioning with 0.1M Glycine-NaOH pH12 with 0.3% Triton X-100. The two injections are a part of the wizard immobilization for C1. I managed to get around 150RU when increasing the concentration to 50ug/ml and running it 60min instead of 7min. So I think 150RU is good enough to work with. Yes, I was thinking of trying the ProA chip definitely, and now I can even compare the data with my C1.

Thanks

SPRofessional