Presentation
Thermal Shift Assay : denaturation / stability of protein with temperature
This q-PCR machine, combined with SYPRO orange dye, is used to mesure denaturation / stability of protein with temperature.
SYPRO Orange binds nonspecifically to hydrophobic surfaces, and water strongly quenches its fluorescence. When the protein unfolds, the exposed hydrophobic surfaces bind the dye, resulting in an increase in fluorescence by excluding water.
The stability curve and its midpoint value (melting temperature, Tm also known as the temperature of hydrophobic exposure, Th) are obtained by gradually increasing the temperature to unfold the protein and measuring the fluorescence at each point.
thermal-denaturation curve
Key words
Thermofluor / SYPRO Orange / q-PCR / stability of proteins / TSA
Staff
Facility manager : Caroline Mas
Operator : Anne-Marie Villard
Office : IBS-room 456
Specific Equipment
CFX connect from BioRad
Access mode
Provision of the instrument after training.
Provision of the instrument is free of charge.
Users have to come with their consumables :
- Sypro Orange (Ref. : S6650 from Molecular Probes – Life technologies).
- PCR plates or strips of any reference. If you use plates, take the “microseal ‘B’ PCR plate sealing film, adhesive, optical” recommended by Biorad (ref. BioRad MSB1001)
Location
Platform : IBS-room 419
How to make a request
Short training on the conditions for provision of the instrument will be done on place (30 min) and is required for the booking. Experimental protocol for the preparation of sample is presented.
• To make a BOOKING REQUEST, fill the agenda in (next to the instrument)
• To make a TRAINING REQUEST : send an email to Anne-Marie Villard
Samples
Only non-pathogenic biological samples are accepted. Decree of the July 18, 1994 establishing the list of biological pathogens, amended by Decrees of April 17, 1997 and June 30, 1998 (Decrees in French). The list of biological pathogens is available on the website of the IPBS.
Follow up / acknowledgements
The user agrees to promote the obtained results by mentioning the platform in the acknowledgements in case of publication or scientific communication and to communicate the reference of the article to the operator and the responsible for the platform.
Acknowledgements in publications :
Please find below the sentence that has to be written in all publications based on our UAR platforms :
« This work used the platforms of the Grenoble Instruct-ERIC center (ISBG ; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). We thank Caroline Mas for assistance and/or access to the biophysics platform. »
