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Protein A amine coupling to sensor chips
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Protein A amine coupling to sensor chips

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1 year 7 months ago #1 by SPRofessional
Protein A amine coupling to sensor chips was created by SPRofessional
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

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1 year 6 months ago #2 by SPRofessional
Replied by SPRofessional on topic Protein A amine coupling to sensor chips
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

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1 year 6 months ago #3 by Arnoud
Replied by Arnoud on topic Protein A amine coupling to sensor chips
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
 

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1 year 6 months ago #4 by SPRofessional
Replied by SPRofessional on topic Protein A amine coupling to sensor chips
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

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