Protein Thermal Shift™ Solution Using Applied Biosystems Real-Time PCR Systems
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The Protein Thermal ShiftTM technology assesses protein stability by measuring changes in melting temperature (Tm) as proteins are heated, using a dye that fluoresces upon binding to exposed hydrophobic regions. This technology enables high-throughput screening of protein stability, ligand binding, and buffer conditions, requiring minimal sample amounts and no prior structural information. The complete solution includes instruments, reagents, and analysis software, making it a valuable tool for protein research and development.
Protein Thermal Shift™ Solution Using Applied Biosystems Real-Time PCR Systems
- 1. Protein Thermal Shift™ Solution Using Applied Biosystems Real-Time PCR Systems Levente Egry Product Manager Real Time PCR Systems
- 2. How does the Protein Thermal Shift ™ technology work? | Life Technologies | 01/13/12 The protein unfolds as it is heated. An environmentally-sensitive dye binds exposed hydrophobic regions and fluoresces. The Tm (melting temperature) is calculated from the melt curve. Changes in Tm are correlated to changes in protein stability. Fluorescence Temperature ( o C) Calculate the inflection point of the curve
- 3. Protein Thermal Shift ™ applications Protein Stability Screen: improving protein preps (pH, salt, excipients) profiling crystallization conditions protein formulation and storage buffers effect of mutations or modifications Protein prep QC High-Throughput Ligand Screening: Small-molecule and fragment screens Antibody-target specificity Protein-protein interaction Inhibitor binding | Life Technologies | 01/13/12
- 4. Protein Thermal Shift TM Solution Advantages of the Protein Thermal Shift ™ Solution : High Throughput: 384 assays in < 30 minutes, robotics available Low sample requirements (usually <1 ug protein / assay) No a priori protein or ligand structure information necessary Minimal upfront optimization | Life Technologies | 01/13/12 Protein Thermal Shift™ Analysis Software Protein Thermal Shift™ Starter and Dye Kits AB Real Time PCR Instruments (proteins from customer) + Reagents & Software enabling a new application for AB ® qPCR instruments
- 5. Protein Thermal Shift™ basic workflow - Calibration | Life Technologies | 01/13/12 Protein Thermal Shift™ software will otherwise not accept *.eds files Each Analysis Group contains a single Reference sample group Use ROX ™ Detector, No Passive Reference Run melt experiment on AB qPCR Instrument Analyze the experiment on the qPCR instrument sw Analyze melt curves in Protein Thermal Shift™ software Open or Start Study in Protein Thermal Shift ™ software Import .eds file(s) into Study A Study is a collection of runs from a single instrument platform Studies can contain >100 *.eds run files
- 6. Protein Thermal Shift™ Software v1.0 Fluorescence and Derivative plots | Life Technologies | 01/13/12 ROA Boundaries Fluorescence Plot Derivative Plot Boltzmann Tm (TmB) Derivative Tm (TmD) Boltzmann Fit Curve
- 7. Drug Screening/Ligand Binding Application To assess shift of the unfolding temperature of the protein tested in presence of ligand relative to that obtained in the absence of ligand. Ligand used: ATP (0.0, 0.1, 1mM) Protein used: T4 DNA Ligase (0.1 mg/ml) DNA Ligase involved in DNA repair and replication ATP is required for the action of DNA Ligase 100mM Potasium Phosphate, pH 6.0, 150 mM NaCl | Life Technologies | 01/13/12
- 8. | Life Technologies | 01/13/12 Protein-Ligand Binding: Increase in Protein Thermal Stability with bound Ligand Protein + Ligand Protein No Protein Control 41 o C 48 o C ViiA TM 7 Real Time PCR System ~ 7 o C delta Tm Fluorescence Melt Curve Derivative Curve
- 9. Protein-Ligand Binding: Increase in Protein Thermal Stability with bound Ligand | Life Technologies | 01/13/12
- 10. Buffer Screening Application Material Tested, RecA (2mg/ml) -> New England BioLabs (M0249L) E. coli RecA is necessary for genetic recombination, reactions involving DNA repair and UV-induced mutagenesis Screening for appropriate buffer conditions to convey stability for protein storage and/or additional test conditions Four buffers are tested for the assay Each Buffer titrated with different concentration of NaCl Sixteen different buffers in total | Life Technologies | 01/13/12
- 11. Buffer Screening Application Sodium citrate pH 5.5 Potassium phosphate, pH 6.0 Potassium phosphate, pH 7.0 Hepes. pH 7.5 | Life Technologies | 01/13/12 0mm NaCl 50mM NaCl 100mM NaCl 150mM NaCl
- 12. | Life Technologies | 01/13/12 Effect of Buffer Conditions on Protein Thermal Stability Buffer Condition Buffer Condition -Na citrate pH 5.5 + 150 mM NaCl -KPO 4 pH 6.0 + 150mM NaCl -KPO 4 pH 7.0 + 150mM NaCl -Hepes. pH 7.5 + 150mM NaCl
- 13. Effect of Buffer Conditions on Protein Thermal Stability | Life Technologies | 01/13/12
- 14. Mutation Screening Application SuperScript® II Reverse Transcriptase (RT) is an improved version of SuperScript® RT (M-MLV) SuperScript® II contains point mutations in the RNase H active center for reduced RNase H activity SuperScript® II mutations within the RNase H domain maintain full polymerase activity Full enzyme activity remains at 42°C SuperScript® III has further mutations for added thermostability | Life Technologies | 01/13/12
- 15. Effect of Point Mutations on Protein Thermal Stability | Life Technologies | 01/13/12 Protein Thermal Shift™ data from ViiA TM 7 instrument showing the Fluorescence and Derivative Melt profiles
- 16. Effect of Point Mutations on Protein Thermal Stability | Life Technologies | 01/13/12 Protein Thermal Shift™ data from ViiA ™7 instrument showing the Fluorescence and Derivative Melt profiles for M-MLV, SSII, SSIII (RED curves) plus ligand (BLUE curves, DNA oligonucleotide)
- 17. Antibody Binding increases Protein Thermal Stability | Life Technologies | 01/13/12 ViiA™7 Real Time PCR Instrument
- 18. Protein Thermal Shift™ Dye Stability | Life Technologies | 01/13/12 Lysozyme incubated at Room Temp in the dark for up to 24hrs with PTS dye. Run under standard conditions on the ViiA™7 at 0.05˚C/sec. ΔTm ≤ 0.34 º C over 24 hours, standard errors of ≤0.04 for each set of 24 replicates per time point. Run on a ViiA™7 Real-Time PCR Instrument.
- 19. Conclusions The Protein Thermal Shift ™ Assay is a rapid, inexpensive, and straight-forward high-throughput tool for screening conditions that maximize protein stability or libraries of ligands. Advantages: Adjustable ramp speed and accurate temperature range flexibility. Small reaction volumes, providing fast and accurate results with < 1µg of protein. Protein TSA has been performed on the Applied Biosystems 7500 Fast, StepOnePlus ™ , and ViiA ™ 7 Real Time PCR Instruments, expanding the flexibility of these systems to protein research. Applied Biosystems’ complete Protein Thermal Shift ™ solution covers the whole range from dye reagent to instrumentation and analysis software. Trial software download at www.appliedbiosystems.com/proteinmelt | Life Technologies | 01/13/12
- 20. For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. © 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. | Life Technologies | 01/13/12 Acknowledgements Carole J. Bornarth Mousumi Rath Nivedita Majumdar Madeline O’Donoghue Junko F. Stevens Marie-Pierre Gauthier Kyle Gee Applied Biosystems, part of Life Technologies Corporation, 850 Lincoln Center Drive, Foster City, CA 94404
Editor's Notes
- #12: We tested 4 different buffers, mostly differing in pH, as well as 4 different salt concentrations for each buffer. Totaling 16 test conditions.