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unstable baseline

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3 months 3 weeks ago #1 by Maria
unstable baseline was created by Maria
Hello, 
i would like to measure the binding affinity between a protein (18 kDa) and some peptides (1-2 kDa). For the experiments i am using a CM5 Chip and a Biacore 3000. I immobilized StrepTactin as capture molecule on the surface via NHS/EDC activation and then captured the protein which was N-terminal modified with a TwinStrep tag. As running buffer i am using PBS (pH 7.4) with 0.1 % Tween.
After some initial test injections i obtained a weird sensogram on the next day as you can see in the attached picture. The baseline shows after some time kind of a jump downwards while only running buffer is used. I also tried some injections with running buffer and one of the peptides which is known to have a good binding affinity, but surprsingliy i got a negative response. 
Now i can´t explain this behaviour to myself. If some Analyt have been left from the first tests and now dissociates slowly, i should´ve noticed by uncomplete recovery. I´ve seen two papers where the same buffer is used for the protein. so this also shouldnt be the problem. 

Thank you for help and greetings,
Maria
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3 months 3 weeks ago - 3 months 3 weeks ago #2 by Arnoud
Replied by Arnoud on topic unstable baseline
Hi Maria,
For me this looks as if the pump stroke is not ok. Check if there is no air in the syringes. Is the dip every time after the pump-fill or not? Did you use the prime after changing the filtered and degassed flow buffer?
A slow dissociation does not look like this, and the dip is quite substantial.
The negative injection signal may point to some blocking in the injection port. Please check if the tubing to the needle is correctly fastened and the injection block is properly connected. You can run unclog to see if that will help.
Kind regards
Arnoud
Last edit: 3 months 3 weeks ago by Arnoud.

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3 months 3 weeks ago #3 by Maria
Replied by Maria on topic unstable baseline
Hi Arnoud,

thank you very much for your answer. Yes i´m always using the prime after docking a new chip or changing the buffer and my buffer is sterile filtered and degassed. Anyways i tried some things after your reply.

First i checked the injection port and cleaned it. Then i also ran unclog. There seems to be no air in the syringes. This nothing changed.

I also checked if the same phenomen occurs with another chip which i used before (same protein, but immobilized via amine coupling). There i coulnd´t recognize this drift, so it seems to be a problem with that specific chip.

So i retried the capturing of my protein on the same flow cell, this time with less flow and longer incubation time. The next day the baseline was fine. I tried some test injections with the running buffer, one of the peptides which is known to have a binding and the antibody. The injection of the antibody led to negative peaks (attached file: Test_150222023). 

Today the drift of the baseline again appeared (attached file: Test_16022023). The response is also higher when it was in the end of the last sensogram. 

Maybe i should also mention that i also immobilized StrepTactin on the reference cell and just omitted the capturing of the protein. The chip is stored at 4°C in a Falcon with a little bit of the running buffer. 

In my opinion something happens with the StrepTactin during the storage. Do you have any experience with that system of iba lifescience?

Thanks and Greetings,
Maria
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3 months 3 weeks ago #4 by Arnoud
Replied by Arnoud on topic unstable baseline
Hi Maria,
I have no experience with StrepTactin on a sensorchip but it is strange that this is occurring for this specific sensorchip. It is good practise to wash the sensor surface with flow buffer and dry both sides (don’t touch the immobilized side). I check both sides visually if they are clean (no deposits, dust, or something).
We had once a strange behaviour due to a little barely visible particle waving in the flow buffer interfering with the response. Sometimes it was there and sometimes not. It could be something like this.
If you run just the flow buffer, can you find a pattern in when the dip occurs? For instance, at 10 ul/min after 3 minutes and at 30 ul/min after 1 minute? Kind regards
Arnoud

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3 months 2 weeks ago #5 by Maria
Replied by Maria on topic unstable baseline
Hi Arnoud,

After the storage i´m drying the chip with some airflow of nitrogen or argon, but i could try to wash it before with some running buffer.

Yes, i can see a pattern. I just checked the sensograms and it seems to be a specific time between the jumps which is shorter with higher flow rate.

I wanted to check if the Tactin causes that phenomen by immobilizing the protein via amine coupling on another flow cell on the same chip. But already the pH scouting was strange, i obtained negative response (see attached file). The positive signals are injections with 50mM NaOH. I already performed the same procedure with the last CM5 chip and everything went fine the last time. The only difference is that the protein is now modified with the TwinStrep tag at the N-terminus.

Best regards,
Maria
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3 months 2 weeks ago #6 by Arnoud
Replied by Arnoud on topic unstable baseline
Hi Maria,

I don't see a problem. Please look at www.sprpages.nl/immobilization/pre-concentration and compare your pre-concentration injections. The negative jump is due to the low salt concentration. Then the protein starts to accumulate on the sensor surface. At pH 4.5 there is probably enough pre-concentration effect to to an immobilisation. The only concern is that the protein seems to stick to the dextran surface. The NaOH washes it away.

I suggest to try an immobilization at pH 4.5 with a slichtly lower ligand concentration than in your figure.

Kind regards
Arnoud

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