Forum :: Bulk change due to the change of RI


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These are the posts of the old forum. It was not possible to transfer the user data, so they are missing in most of the posts. For new questions, go to the general discussions.

TOPIC: Bulk change due to the change of RI

Bulk change due to the change of RI 19 Mar 2013 01:00 #1

Hi,

I am doing SPR experiment using a homemade setup. I have a bulk change problem during the exchange of solutions. This is what I did: PBS-water-2 propanol-water-PBS. According to my understanding, these solutions will not bind to the surface of the chip. So after the whole process, I should get back the baseline level. However, I observed a change in baseline level at the end. I can see a lot of published results with the commercial SPR machine are showing the same problem. May I know who can help me to explain what is happening? Thank you very much.
weiru

Bulk change due to the change of RI 19 Mar 2013 01:00 #2

Hi,

You are correct that the solutions should not bind. What you not say is what surface you use in the experiment. In case of a dextran matrix you can observe some baseline shift due to vertical shrinking or extension of the matrix. I expect that the 2 propanol has the largest effect.

Regeneration solution like high or low pH, and high salt also can change the base line. In addition, bound proteins can also change by this solutions.

I know that in all these cases there is no mass change on the sensor chip surface. However i think that somehow the density of the surface alters the refractive index of the system and therefore the baseline.

Can you tell what the differences where?

Arnoud

Bulk change due to the change of RI 19 Mar 2013 01:00 #3

Hi,

Thanks for the reply. I have a carboxylated surface with 16-MHA (16-mercaptohexadecanoic acid).

I have a similar baseline difference with NHS/EDC. The difference that I am talking about: the power measured for a bound protein is 0.3dB, the difference in power after flowing NHS/EDC is 0.2dB.

Bulk change due to the change of RI 19 Mar 2013 01:00 #4

I am not familiar with this read out, so I cannot comment if it is high or low.
The 16-MHA has a free -COOH group. The NHS/EDC can activate the -COOH group to N-hydroxysuccinimide ester. This is slightly bigger so will give a positive signal. The ester will detoriate over time.