Positive RU values appeared Blank channel

Hellow,

I'm Biacore T200 user and have been using Biacore T200 for a month.

I have a problem with my analysis, so I need some advices.

I was used two channel

Fc 1 : Blank channel to substract main analysis channel.
Fc 2 : Main analysis channel.

In my sensorgram, Fc 1 channel have significant rise of RU value during sample injection.

The first cycle does not seem to be a big problem, but as the analysis progresses, the sensorgram RU value increases significantly in Fc 1 channel.

Not only Fc1 but also Fc2 value increases gradually as the analysis progresses.

Both FC1 and FC2 tend to increase RU gradually, however, baseline does not seem to have much difference during each cycle.

When Fc1 is subtracted from the Fc2 value, the maximum RU value gradually decreases and the baseline gradually decreases.

After a certain period of time after end of analysis, the first analysis has little difference in Fc1, but the same phenomenon occurs as the analysis is repeated.

I would like to attach a picture, but it is difficult to attach it directly to the situation, but if you need a picture, I will try it.

Thanks for reading, and I hope any advice about this problem.

Thanks,

Hi,

Welcome to the SPR community.

It is difficult to understand from your description where exactly the rise is (during the analyte injection or between cycles). However, you can try several things to make the instrument more equilibrated and stable (I suggest you read www.sprpages.nl/best-results ).

To check that the system is stable and well equilibrated you can run several dummy cycles (3 – 6), injecting flow buffer only. You will see that the first cycle will be significantly different from the rest. The reason is that the sensor chip has to 'set' for this new condition. When the channels are equal, subtracting ch2 – ch1 should give a flat line for ch2.

Then run some cycles injecting flow buffer with some added salt (+25 mM NaCl). This wil give an injection block (also referred to as bulk injection jump). Depending on the ligand immobilized on the channel there can be a difference in extend of the jump. However, reference subtraction and double referencing should compensate for the differences.

Maybe you can show one or two figures showing your problem.

Kind regards

Arnoud

To Aroud

Thank you for your kind advice, and I am sorry I did'nt explain my problem in detail.

I read the information you gave me, and I also perforemed dummy cycle what you suggested.

Dummy cycle was performed using running buffer (HBS-EP+) and running buffer was administered five times in the manual run (total 5 cylce).

Only the running buffer was flowed in both the Fc1 and Fc2 channels, but the shape of the sensorgram with the box shape appeared (Box shape sensorgram means that the RU value rises to the highest level as soon as running buffer is injected, and drops to the original level when the injection is discontinued).

As mentioned in the above question, I have confirmed that the RU value is higher in the second than in the first, and in the third run rather than the second.
The following is an real data of the RU value measured

Report point is 5 second before end of injection.


Fc 1 channel

Cycle 1 : 1.2 RU
Cycle 2 : 5.4 RU
Cycle 3 : 10.2 RU
Cycle 4 : 15.8 RU
Cycle 5 : 19.4 RU

I would appreciate it if you let me know what you think about this situation
I think there is more problem in my experiment (or Biacore instruments).

I want to hear any advice or recommandation for this problem.

Thanks for reading!

Hi,
Do you use the caps on the vials?

To Arnoud

I have not been instructed to use cap on vials, so I am not using it now.

Could this be the cause of the problem?

The caps prevent evaporation of the solution. Evaporation will give a higher buffer concentration and since SPR is measuring (bulk) mass it will give a higher response. Because the responses are higher each cycle it is possible that this is due to evaporation. 1 mM NaCl difference give ~ 20 RU!