Forum :: Negative RU upon analyte injection


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This forum is intended for questions about kinetics, Surface Plasmon Resonance and the instruments related to these techniques.

TOPIC: Negative RU upon analyte injection

Negative RU upon analyte injection 10 Mar 2016 08:37 #1

Hi,

I am monitoring hIgG-antigen binding kinetics using Biacore T200. I use the CM5 chip with human antibody capture kit from GE. The regeneration buffer is 10 mM Gly-HCl. I made a brand new chip and performed several measurements (less than 200 cycles) within one week. Everything was fine.

Today I completed another set of experiment using the same chip. I analyzed the data using BIAevaluation and the sensorgrams all looked fine. But when I take a look at the real sensorgrams I found a weird thing that the RU actually decreased upon analyte injection. This phenomenon can be observed for both startup cycle (human IgG isotype control with HBS-EP buffer) and the samples.

negativeresponse-1.png


I looked at the previous results I had just a few days ago and the RU did increase upon analyte injection. The baseline did not drift, so after double referencing the relative RU just looked fine, and I had no problem getting good fitting.

negativeresponse-2.png


I don't know if this would be a potential problem that I have to make a new chip just to be on the safer side. I also wanted to mention that two weeks ago I had some nonspecific binding on another chip when injecting HBS-EP buffer only. It turned out that the buffer I used may be contaminated (filtered and stored at 4 C for three months). I cleaned the instrument (desorb and sanitize), made another chip and confirmed that the instrument was clean and the chip was fine with new buffer and experiments.

Should I make another new chip or I can continue to use this chip since it is pretty new. Or this is the problem of change of refractory index upon injection which does not matter.

Thanks!
Last Edit: 10 Mar 2016 09:16 by yesucan.

Negative RU upon analyte injection 11 Mar 2016 09:07 #2

Hi,

First a note on buffers. It is better to make buffer fresh (as you experienced). In general we store buffer for one weak and prepare fresh filtered water every day. It takes only five minutes and will keep the instrument clean.

The negative buffer jump looks more a buffer mismatch.

If you connect the buffer to the instrument do you use the cap and is it tightened? Without a cap the buffer will evaporate and the salt concentration will increase and thus giving a higher baseline. Since your sample is probably diluted with the original buffer it has less salt en thus upon injection you get a negative signal (1 mM NaCl ~ 20 RU).

If double referencing is compensating for the negative jump I should not make a new chip.

Regards

Arnoud

Negative RU upon analyte injection 14 Mar 2016 02:29 #3

Hi Arnoud,

Thanks for the advise on buffers. I did use cap for the bottles, though. After making everything fresh from the start there's no more buffer mismatch. It's also good to know that salt content may contribute to change of RU.

Thanks again,

yesucan

Negative RU upon analyte injection 04 May 2016 01:29 #4

I have to agree with Arnoud here. This is just an issue with an index match with the buffer, and is not a problem if you are employing double referencing. The baseline will also decrease in the control channel and then can be subtracted from your active channel. Any buffer blanks will further correct the data (and of course complete the data set).
Thanks!
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