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

When proteins matter. Dr. Mathilde Belnou | Technical Sales Specialist Confidential. Do not distribute.

1856 / 1879 2008 2010 2012 2014 2016 2017 2018 2019 Monolith Monolith Solutions for Foundation: Tycho NT.6 NT.115 NT.Automated full automation LMU Munich Prometheus Carl NT.48 Ludwig Dianthus NT.23 Red Dot Growth Fast thermo- Design Award Champion optical particle characterisation PCT Patent 2008/061706 Charles Top 100 – Germanys German Soret most innovative Innovation Award company German Founders Prize Confidential. Do not distribute.

When binding affinity matters. Microscale Thermophoresis (MST) Monolith Product line Confidential. Do not distribute.

An exampleofMST in action DNA Duhr and Braun, PNAS, 2006 Duhr and Braun, PRL, 2006 Confidential. Do not distribute.

TRIC and Thermophoresis contribute to measured MST signal TRIC + MST signal = Thermophoresis Laser Laser “off“ “on“ Both TRIC and thermophoresis are influenced by binding events and therefore contribute to the overall recorded MST signal Baaske et al., Angew Chem Int Ed Engl , 2010 Confidential. Do not distribute.

This ishowMST works Confidential. Do not distribute.

What does the data tell me about the binding interaction? F 0 F 1 K d bound Fit unbound Confidential. Do not distribute.

Theory of thermophoresis: contributing factors Size Hydration Shell Charge 2 Duhr and Braun, PNAS , 2006 Duhr and Braun, PRL , 2006 Confidential. Do not distribute.

Interactions accessible with MST Confidential. Do not distribute.

MST technique, Monolith device • No maintenance • Easy-to-use • Solution • Few µL (K D = 160 µL [10 nM] of labelled protein) • Work in complex bioliquids • 10 min • Affinity range : pM – mM • Any kind of biomolecular interaction • Confidential. Do not distribute.

Applications Confidential. Do not distribute.

Protein dimerization Grb2 dimer • 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 Confidential. Do not distribute.

Ca 2+ binding to synaptotagmin Adapted from van den Boogaart et al. • Detect even smallest binding induced changes – also label-free Max Planck Institute for Biophysical Chemistry and University Göttingen, Germany The Journal of Biological Chemistry, 2012 Confidential. Do not distribute.

Nitrate binding to a 12 transmembrane-helices transporter • Measure the unmeasurable: Detect smallest binding induced changes – even for difficult targets. Adapted from Parker & Newstead, University of Oxford, UK Nature, 2014 Confidential. Do not distribute.

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 Confidential. Do not distribute.

Competition experiments for p38 inhibitors Material was kindly provided by Dr. Krishna Saxena, • Easy screening for competitive and allosteric inhibitors University Frankfurt, Germany Confidential. Do not distribute.

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 Confidential. Do not distribute.

There is a reproducibility crisis in research Nature 533, 452 – 454 (26 May 2016) Confidential. Do not distribute.

“I’m just following the protocol the way I was shown when I joined the lab….” Wikimedia commons Confidential. Do not distribute.

Common tools researchers use for protein characterization Concentrati Size and Separation and on and molecular resolution purity weight Confidential. Do not distribute.

Quick results on protein quality, label-free and using little sample Confidential. Do not distribute.

Quick results on protein quality, label-free and using little sample Results in 3 minutes 35°C ➔ 95°C 30°C/min Confidential. Do not distribute.

Quick results on protein quality, label-free and using little sample Results in 3 minutes 35°C ➔ 95°C 30°C/min Only microliters of sample required 10 µL 10 µg/mL ➔ 200 mg/mL Confidential. Do not distribute.

Quick results on protein quality, label-free and using little sample Label-free No immobilization Results in 3 minutes 35°C ➔ 95°C 30°C/min Only microliters of sample required 10 µL 10 µg/mL ➔ 200 mg/mL Confidential. Do not distribute.

nanoDSFtechnology Dual UV-detection • Rapid detection of trytophan fluorescence without losing unfolding information Confidential. Do not distribute.

What Tycho informs you • Inflecti ction n tempe mperature rature (T i ) • Initi tial rati tio • Δ ratio • Sample brightness • Profile similarity (%) Confidential. Do not distribute.

Fast evaluation of oxidation-induced destabilization of mAbs Oxidation of mAb results in reduced binding capabilities Confidential. Do not distribute.

Better optimization of biosensor assay conditions pH 5.5 recommended for immobilization Confidential. Do not distribute.

Quick protein binding tests by label-free thermal shift analysis Confidential. Do not distribute.

Efficient ID of target protein in chromatography fractions Easily compare profiles after each run to determine similarity Confidential. Do not distribute.

Monitor theeffectsofstorageconditionson proteinquality Confidential. Do not distribute.

Thank you for your attention. mathilde.belnou@nanotempertech.com | support@nanotempertech.com Confidential. Do not distribute.