Forum :: How Preconcentration Can Save Your Protein Samples in SPR Exper


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

TOPIC: How Preconcentration Can Save Your Protein Samples in SPR Exper

How Preconcentration Can Save Your Protein Samples in SPR Exper 21 Apr 2016 22:11 #1

Hey everyone!

I thought of sharing one of my favourite SPR tips - preconcentration! This technique basically allows researchers to use even less ligand when conducting SPR experiments. Since expressing and purifying proteins is time-consuming and purchasing proteins can get expensive, saving as much ligand as you can is always ideal.

Even a lot of our own users were asking us to explain preconcentration, so we put together a blog post to help out. We hope you enjoy. :)

Blog link: www.nicoyalife.com/blog/preconcentration...e-plasmon-resonance/
Learn more about OpenSPR, the world's first SPR for individual researchers, at www.nicoyalife.com

How Preconcentration Can Save Your Protein Samples in SPR Exper 23 May 2016 13:17 #2

Hi there, thanks for the interesting blog post!

You have stated that peptides and small molecules are unsuitable ligands for pre-concentration, I was just wondering why this is? I have recently been trying both and the results have seemed odd to me so it would be nice to know whether I am wasting my time needlessly trying to optimise conditions.

Also, in the case of these unsuitable ligands is it best to simply perform immobilisation in running buffer instead?

Best regards,

Tom

How Preconcentration Can Save Your Protein Samples in SPR Exper 24 May 2016 01:38 #3

Mr. Bigwell,
This article is describing advantages of the technique of pH scouting. By placing a ligand in an array of pH conditions, you can minimize the amount of ligand used for successive experiments. As stated, it is very useful for expensive or difficult to purify proteins. See link for an example of pH scouting:

https://www.researchgate.net/figure/223965303_fig1_Fig-1-A-Curves-of-the-pH-scouting-experiments-showing-that-10-mM-acetate-pH-40

Note that the increase in the SPR signal (Response) is due to a change in the mass on the surface of a sensor. When you use a protein, enzymes or other molecules that are 5kDa and above, it is much easier to detect the rate at which these ligands are preconcentrating on the surface, but when the molecular weight falls to a few hundred Daltons or below, it is very difficult to decern. It's not that it doesn't work, its that it is difficult to detect and choose the appropriate pH. Hope this helps!
Thanks!
The following user(s) said Thank You: Bigwell

How Preconcentration Can Save Your Protein Samples in SPR Exper 24 May 2016 07:05 #4

That makes sense and seems to explain some of my results which I had previously questioned, thank you for the explanation!
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