CM5 chip issues

Dear All,
good day. Hope you're doing well and stay healthy.
I am having problems working with the CM5 chip. Is there anyone who can help and give advice?
1) Immobilization of streptavidin: in which buffer can it be diluted? I tried on a running buffer where I got a low bind response. Then tried to use acetate buffer, which turned out to be below the baseline.
2) At the injection stage, everything goes right at the beginning. And when it comes to the stage of stability, RU is sharply reduced. Became less than initial baseline.
3) What to do if during the system check in Biacore X100: noise or response is failing?
4) If possible, can someone who can help me give an email, so I want to send the graphs and discuss further.

Hi,
1) Protocol: Immobilization of streptavidin.
Activate a carboxymethylated dextran surface with standard NHS/EDC activation.
Inject at 5 µl/min, 40 µl of streptavidin [100 µg/ml in 10 mM NaAc, 0.1 mM EDTA, 1 mM NaCl, 1 mM DTT pH 4.6] over the activated surface.
After coupling, deactivate the surface by injection of 35 µl of 0.1 M ethanol-amine, pH 8.0.

Protocol: capturing of biotinylated protein.
• Inject a biotinylated protein of your choice (e.g. 50 µl of 50 µg/ml IgG in running buffer) over the immobilized streptavidin.
• Unbound proteins can be removed with a pulse of 2 M NaCl at 20 µl/min.

When making a reference surface on the streptavidin sensor, it is not sufficient to activate and deactivate the surface. The best practice is to immobilize the same amount of streptavidin and to quench this with D-biotin, to make the surfaces better comparable.

2) not sure what you inject at this stage...
3) best is to use fresh running buffer and equilibrate longer before attempting the system check.
4) you can post sensorgrams in the forum.

Kand regards
Arnoud

Dear Arnoud,
thank you a lot!
in the case of 2, it can occur at anytime, after injection of streptavidin or our Aptamer and etc.

Next thing that I want to ask is What if in the system check process, the "response" or noise" is failing?

Thank you.

Hi,

I am not exactly familiar with the X100 but the first thing you can do is to compare a previous correct system check with the failed one. This can give you clues what the difference is and what exactly is failing. Too high noise (and drift) can come from dirty instruments or not well equilibrated instruments. Also check the tubing, injection port and needle. Is the response too high or too low? This problem can come from the test solution which is not correct. It can be difficult to make this solution in the lab and have correct readings from the instrument.
Second you can test the instrument yourself with running buffer and NaCl. Make a dilution series of running buffer with +500 mM NaCl and dilute until ~+15 mM NaCl (+ running buffer alone) and inject 1-2 minutes over a fresh well equilibrated CM5 sensor chip. The curves should have a sharp rise and fall and the response proportional to the NaCl concentration. If not you have to contact your maintenance provider.
Kind regards
Arnoud