October 17, 2021, 08:35:37 PM
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Topic: Surface Plasmon Resonance Woes  (Read 271 times)

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Offline cplesica

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Surface Plasmon Resonance Woes
« on: September 20, 2021, 03:50:15 PM »
Hello everyone, I am looking for an expert (or at least someone that knows more than I do) about surface plasmon resonance (SPR).

I am trying to study an interaction between a protein and a lipid using this technique. By two other techniques (lipid overlay and lipid sedimentation) I have shown that my protein does interact with my lipid of interest. I want to quantify this interaction with SPR.

I have a positive control protein, known to bind the lipid with high affinity, and a negative control, inert protein which should not interact with my lipids.

I have made 100 nm liposomes containing my lipid of interest, immobilized them on a L1 chip, and obtained the readings attached in the graph here.

My question is: why does my test protein not look anything like my positive or negative controls? What could be causing the shape of the curves, the huge negative dip at the beginning of the run, and the large number of negative RUs.

Everything is in the same SPR running buffer except the negative control protein (in water)... HEPES and NaCl pH8.

Anyone ever seen anything like this or could offer some advice?

Thank you!

Offline Corribus

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Re: Surface Plasmon Resonance Woes
« Reply #1 on: September 21, 2021, 11:14:05 AM »
Could you provide a little more about your exptl procedure?

It seems you are binding liposomes to the surface first (in a monolayer?), then looking to see whether your protein binds to this monolayer. Your positive control looks right. Is it possible your test protein is disrupting the lipid monolayer - i.e., your monolayer is dissociating from the surface, leading to an initial loss in RU?
What men are poets who can speak of Jupiter if he were like a man, but if he is an immense spinning sphere of methane and ammonia must be silent?  - Richard P. Feynman

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