Forum :: RU increase upon injection finish


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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.
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TOPIC: RU increase upon injection finish

RU increase upon injection finish 08 Apr 2011 02:00 #1

Hello All,

Firstly, thanks for getting this site set up, it has been especially handy since I've been self-taught SPR and will be getting the book soon.

I'm currently using a BIAcore X with HBS-N + EDTA (4.0mM) as running buffer. Ligand is a glycoprotein coupled to a CM5 chip by amine chemistry.
I've noticed that the sensorgram response (in terms of RU) increases sharply (in a manner dependant on sample concentration) immediatly after the injection of analyte ends.
I'm concerned that the IFC may have a fluidics delivery issue, but I wonder if there may be another cause to this effect as injection of other solutions (BIAtest and antibodies against my immobilised ligand) appear to behave normally.
I had thought that non-specific binding to FC1 may have caused delayed binding to FC2 but the non-specific binding is always around 15 RU (my analyte responces are typically +1000 RU (since I'm doing steady state analysis at the moment)) and the jump after injection varies from 60-200+.
I'm wondering if anyone has seen this occur, perhaps there is a way of working around it?
There is one other curiosity I have: the analyte (a phosphorylated glycolipid) appears to have a negative refractive index (stock is in pure water, but this happens when diluted in running buffer and seems dependant on analyte concentration), does that seem strange?

I would really appreciate any feedback,

Best wishes,

James

RU increase upon injection finish 08 Apr 2011 02:00 #2

You are sure it is not a bulk RI change when going back to running buffer? This would agree with the concentration dependence (if the jump is proportional). But I guess that you also referencing this, right?
joseantonio

RU increase upon injection finish 08 Apr 2011 02:00 #3

Cheers for the response joseantonio.

Aye, the change in RU seems to exceed the RI change between sample and running buffers. I hope its not a fluidics issue, perhaps it is due to the lack of surfactant (P20) in running buffer?
james

RU increase upon injection finish 08 Apr 2011 02:00 #4

You should definitely add surfactant to your running buffer. Can you tell the bulk RI change from that other RI change you mention? If so, how do you do that? Have you tried injecting just running buffer?

RU increase upon injection finish 08 Apr 2011 02:00 #5

Problem is P20 prevents the interaction between my analyte and ligand. As it goes I am using it for Regeneration solution (it works a treat on hydrophobic interactions). The dips either side of the injection result from the delay between RI changes in FC1 and FC2 and they match up in terms of RU change. The raise in RU that I'm seeing post injection is considerably more though. But yes we also injected running buffer only as well, which correlated nicely. I wonder if this issue might be caused by adsorbed aggregates in the IFC?

RU increase upon injection finish 08 Apr 2011 02:00 #6

I apologize but I dont the get picture. Could you post a sketch (made in paint) or a screenshot of the response in FC1 and FC2)?
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