When proteins matter. Dr. Mathilde Belnou | Technical Sales - - PowerPoint PPT Presentation

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When proteins matter.

• Dr. Mathilde Belnou | Technical Sales Specialist

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Monolith NT.Automated 2010 Monolith NT.115 Solutions for full automation 2012 2014 2016 1856 / 1879

Carl Ludwig Charles Soret Foundation: LMU Munich

2008

Fast thermo-

• ptical particle

characterisation PCT Patent 2008/061706 Red Dot Design Award German Innovation Award

Prometheus NT.48

German Founders Prize

2017

Growth Champion

2018 Tycho NT.6

Top 100 – Germanys most innovative company

2019 Dianthus NT.23

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When binding affinity matters.

Microscale Thermophoresis (MST) Monolith Product line

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An exampleofMST in action

DNA

Duhr and Braun, PNAS, 2006 Duhr and Braun, PRL, 2006

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TRIC and Thermophoresis contribute to measured MST signal

Laser “off“ Laser “on“

MST signal Thermophoresis TRIC +

Baaske et al., Angew Chem Int Ed Engl, 2010

Both TRIC and thermophoresis are influenced by binding events and therefore contribute to the overall recorded MST signal

=

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This ishowMST works

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What does the data tell me about the binding interaction?

unbound bound F0 F1

Fit Kd

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Theory of thermophoresis: contributing factors

Size Charge2 Hydration Shell

Duhr and Braun, PNAS, 2006 Duhr and Braun, PRL, 2006

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Interactions accessible with MST

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• Solution
• Few µL (KD= 160 µL [10 nM] of labelled protein)
• Work in complex bioliquids
• MST technique, Monolith device
• No maintenance
• Easy-to-use
• 10 min
• Affinity range : pM – mM
• Any kind of biomolecular interaction

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Applications

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Protein dimerization

• Straightforward detection of multimerization affinities – independent of size

Adapted from Lin et al., Cell, 2012 and Ahmed et al., Nature Comm, 2015 Ladbury Lab, University of Texas, MD Anderson Cancer Center

Grb2 dimer

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Ca2+ binding to synaptotagmin

• Detect even smallest binding induced changes – also label-free

Adapted from van den Boogaart et al. Max Planck Institute for Biophysical Chemistry and University Göttingen, Germany The Journal of Biological Chemistry, 2012

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Nitrate binding to a 12 transmembrane-helices transporter

Adapted from Parker & Newstead, University of Oxford, UK Nature, 2014

• Measure the unmeasurable: Detect smallest binding induced changes – even for difficult targets.

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Interaction of ssDNA with Single-Strand Binding Protein (SSB)

• Measure affinities that are not accessible with other methods!

adapted from Jerabek-Willemsen et al., NanoTemper Technologies Journal of Molecular Structure, 2014

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Competition experiments for p38 inhibitors

• Easy screening for competitive and allosteric inhibitors

Material was kindly provided by Dr. Krishna Saxena, University Frankfurt, Germany

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Analyze difficult proteins without purification

• Labeling + interaction characterization of non-purified proteins in lysate
• Method exclusive to NanoTemper

Kleusch et al., NanoTemper Technologies Application Note NT-MO-032, 2017

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Nature 533, 452–454 (26 May 2016)

There is a reproducibility crisis in research

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“I’m just following the protocol the way I was shown when I joined the lab….”

Wikimedia commons

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Common tools researchers use for protein characterization

Concentrati

• n and

purity Separation and resolution Size and molecular weight

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Quick results on protein quality, label-free and using little sample

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Results in 3 minutes 35°C ➔ 95°C 30°C/min

Quick results on protein quality, label-free and using little sample

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Only microliters

• f sample required 10 µL

10 µg/mL ➔ 200 mg/mL Results in 3 minutes 35°C ➔ 95°C 30°C/min

Quick results on protein quality, label-free and using little sample

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Only microliters

• f sample required 10 µL

10 µg/mL ➔ 200 mg/mL Label-free No immobilization Results in 3 minutes 35°C ➔ 95°C 30°C/min

Quick results on protein quality, label-free and using little sample

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nanoDSFtechnology

Dual UV-detection

• Rapid detection of trytophan fluorescence without losing unfolding information

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• Inflecti

ction n tempe mperature rature (Ti)

• Initi

tial rati tio

• Δ ratio
• Sample brightness
• Profile similarity (%)

What Tycho informs you

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Oxidation of mAb results in reduced binding capabilities

Fast evaluation of oxidation-induced destabilization of mAbs

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pH 5.5 recommended for immobilization

Better optimization of biosensor assay conditions

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Quick protein binding tests by label-free thermal shift analysis

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Easily compare profiles after each run to determine similarity

Efficient ID of target protein in chromatography fractions

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Monitor theeffectsofstorageconditionson proteinquality

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Thank you for your attention.

mathilde.belnou@nanotempertech.com | support@nanotempertech.com