Thermal Denaturation Assay - TDA

 
 

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Instrument:


The Biometra TOptical rtPCR system is located in Swann 3.10. It is an RT-PCR machine which has been setup exclusively for thermal denaturation assays.

















Further technical information about this instrument can be found on the Biometra website.

Please click here for individual platform/resource costings.

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Features of the TDA on the TOptical:

  1. 96-Well plate format is used.


  1. Well volumes ≤ 50 μL, ensuring low consumption of materials, including protein and ligands.


  1. Run times can be as short as 25 minutes allowing for high throughput assays.


  1. Wide range of ligand binding affinities can be detected.


  1. Dedicated SYPRO Orange filter set.


  1. 3 SOPs for screening/analysis - Low thermal stability, Standard thermal stability and High thermal stability.


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What is a TDA?

The thermal denaturation assay is a method used to measure the thermal stability of proteins and will directly measure the transition unfolding temperature (Tm). It works by using the fluorescent dye Sypro orange which is environmentally sensitive. As proteins unfold due to increasing temperatures, the dye will be exposed to large hydrophobic regions, resulting in a large increase in fluorescence (ex/em 490nm/580nm). The figure below shows typical traces for this process with given in the form of RFU vs T, and –(δRFU) vs δT. In the trace B, Tm is at the peak minimum, which corresponds to the point of inflexion in trace A.

 


 


 

The TDA can be used in a variety of ways. For instance, it can be used to identify suitable buffers and additives for protein stabilisation (positive shift in Tm) which could aid the crystallisation process, or even improve on existing crystal quality. Secondly it can be used as a primary screen for ligand identification. Again, ligand binding would lead to a positive Tm shift compared to the unliganded protein, and could identify hits or families of compounds that can be taken through to a second round of screening or inhibitor development. The ligand binding affinity can also be estimated from the change in transition melting temperature (ΔTm).


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contacts:


Facilities Manager:

Dr. Martin Wear

Rm. Swann 3.20

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: martin.wear@ed.ac.uk


Protein Technologist:

Dr. Liz Blackburn

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: E.A.Blackburn@ed.ac.uk


Protein Technologist:

Dr. Matt Nowicki

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: matthew.nowicki@ed.ac.uk



Facilities:


CTCB-Home

Protein Production Biophysical Characterisation

In Silico Screening

Training


Links:


On-line Booking

Core Column Library


Access Charges/Costs:


Access Charges/Costs

 

Location:

The University of Edinburgh,

Level 3 Michael Swann Building,

King’s Buildings,

Mayfield Rd.,

EH9 3JR,

UK


Lab 3.10

TDA - TOptical