Can I use 0.1% NP40 (alternate) in running buffer (Biacore 3000)? - Forum


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TOPIC: Can I use 0.1% NP40 (alternate) in running buffer (Biacore 3000)?

Can I use 0.1% NP40 (alternate) in running buffer (Biacore 3000)? 18 Oct 2019 21:17 #1

  • Charlesmsa7
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Hi,
I am using Biacore 3000 machine.
My objective is to study biotinylated peptide and peotein interactions. Am planning to immobilize peptide on SA chip and inject proteins (analyte) over it.
How much maximum percentage of NP40/P20/Tween I can use in the running buffer?.
In my other assays I've optimized the conditions as 0.1%. And all the papers published were reported only 0.05% of surfactant or lesser than that. How surfactant beyond 0.05% will affect the interactions study and SPR signals?
Or
Generally biochemical assays use more amount of surfactant, whereas SPR uses very low. How I can optimize the surfactant quantity?.

Expecting a ultra quick response.
Thank You all in advance
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Can I use 0.1% NP40 (alternate) in running buffer (Biacore 3000)? 21 Oct 2019 20:17 #2

Hi,
the most used detergent concentration is 0.005% tween 20 (P20: polysobate 20). The 0.005% is around 70% of the CMC of Tween-20. The detergent is used to lower the surface tension and to reduce non-specific binding. Sometime higher concentrations are used - most used range 0.005 - 0.02.
I would not use 0.1% NP40 but a lower concentration e.g. 0.01% or even lower to start with.
How the detergent will change the protein interaction you have to find out your self. No common guideline there as far as I know.
Last Edit: by Arnoud.
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Can I use 0.1% NP40 (alternate) in running buffer (Biacore 3000)? 22 Oct 2019 13:50 #3

  • Charlesmsa7
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Dear Arnoud,
You mean to say that the micelles formed due to higher concentration (above CMC) of detergent will affect the SPR sensogram?. Bcoz, whatever the concentrations you are mentioned for P20 and NP40 are lower than CMC.

But the Biacore Assay handbook says the fololwing (Page 24, Section 3.3.3 additives, detergents)

""Inclusion of non-ionic detergent (0.05% Surfactant P20, TweenTM or equivalent)
in all buffers is recommended to minimize deposition of protein and other
biomolecules in the flow system. The detergent should be included at a
concentration above the critical micelle concentration (CMC). At lower
concentrations, the detergent itself can accumulate in the flow system and lead
to unwanted response effects as it is released""

I have gone through more than 70 papers. Most of the papers using 0.005 or 0.05% of tween.
CMC of Tween is ~0.0074 % w/v or ~0.06 mM.

How this can be justified? using lower concentrations than the CMC in general experiments.
Then, How i can determine the optimum concentration of detergent for SPR?. I couldn't find any standar protocol or assumptions for this.
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Can I use 0.1% NP40 (alternate) in running buffer (Biacore 3000)? 22 Oct 2019 20:33 #4

Hi,

Taking the Assay Handbook we all should use at least a detergent concentration above the CMC.
I looked up de buffers sold by GE and this is strange since the ready to use general purpose buffer has 0.005% w/v surfactant P20 but the 10x concentrated stock contains 0.5% thus when diluted 0.05%. Seems that they are also not consistent…

As far as I know the effect of detergent in kinetics is not systematically investigated and published. However the effect of detergents on small compounds was published in:

1. Giannetti, A. M., Koch, B. D. and Browner, M. F.; Surface Plasmon Resonance Based Assay for the Detection and Characterization of Promiscuous Inhibitors. Journal of Medicinal Chemistry (51) 3: 574-580; 2008.
2. Ryan, A. J., Gray, N. M., Lowe, P. N., et al.; Effect of detergent on "promiscuous" inhibitors. J.Med.Chem. (46) 16: 3448-3451; 2003.

Kind regards
Arnoud
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Can I use 0.1% NP40 (alternate) in running buffer (Biacore 3000)? 23 Oct 2019 07:58 #5

  • Charlesmsa7
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Arnoud,
Considering streptavidin chips,

Similar platform BLI (Octet, BioLayer Interferometry) also uses streptavidin coated biosensors. The immobilization pattern and assays are similar for SPR and BLI. They have given maximum/validated contact time of reagents with the BioLayer. Check Table.1 of the following document.

www.biophysics.bioc.cam.ac.uk/wp-content...din_regeneration.pdf

Also they mentioned that the higher contact time may damage the streptavidin surface. Can we possibly correlate with that data with Biacore SA chip stability?

Regards
Charles M
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