Isothermal Titration Calorimtery - MicroCal Auto-iTC200

 
 

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Instrument:


The MicroCal Auto-iTC200 instrument is located in Michael Swann laboratory 3.10.




Overview and technical specifications.

Information on ITC technology and Microcal's products.


Please click here for individual platform/resource costings.

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Features of the Auto-iTC200 instrument:


  1. Direct and label-free measurement in solution, of binding affinity, stoichiometry, enthalpy and entropy.


  1. Fully automated system ensures fast, accurate sample handling and cleaning.


  1. Over 20 titrations per day can be performed, using typical run parameters.


  1. The sensitivity of the instrument is comparable to the iTC200 instrument.


  1. Active* cell volume is 200 µl.


  1. Cell has Peltier-controlled temperature range of 2 - 80 °C.


  1. Separately temperature-controlled sample trays hold up to four 96-well plates and five 30 ml tubes.


  1. Sample recovery can be programmed into the run parameters.

* (Requires 400 µl for sample in cell and 120 µl for ligand in syringe. This is 5-fold less volume than was required for the previous generation of instrument instrument).


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What is ITC?


Isothermal Titration Calorimetry (ITC) is a thermodynamic technique for measuring any chemical reaction initiated by the addition of a binding component, and is frequently utilised for the characterization of biomolecular interactions.  When substances bind, heat is either generated or absorbed.  Measurement of this heat allows accurate determination of binding constants (KB), reaction stoichiometry (n), enthalpy (ΔH) and entropy (ΔS), thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment.  In ITC, the usual experimental set-up has a syringe containing a “ligand” solution which is titrated into a cell containing a solution of the “macromolecule” at constant temperature. When the ligand is injected into the cell, the two components interact and heat is released or absorbed in direct proportion to the amount of binding. As the molecule in the cell becomes saturated with ligand, the heat signal diminishes until only the background heat of dilution is observed. An example of type of data an ITC experiment generates is shown in the figure below.




Calorimetric titration of the undecapeptide drug cyclosporin-A (CsA) with recombinant, human, His-tagged cyclophilin-A (His-CypA) (from Wear & Walkinshaw, 2006). Each peak (upper panel) represents the injection of 5 µl 42.7 µM His-CypA from the syringe into 1.41 ml of 3 µM CsA at 25 °C in 10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA; 1 % ethanol.  Due to the solubility problems of CsA, this was the molecule in the cell.  Heats of reactions were determined by integration of injection peaks with correction for heats of dilution. The resulting titration curve (lower panel) is the least-squares fit to a single-class binding site model using MicroCal Origin software supplied with the instrument. The thermodynamic parameters determined in this experiment are: Kd = 11.5 ± 1.3 nM; ΔH = - 14.6 ± 0.05 kcal/mol, TΔS = -3.8 kcal/mol, n = 1.05 ± 0.01.


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Booking and rules for use of the instrument:


The machine must be booked prior to use.  A web-based booking system is available for registered users at the link below. You cannot book more than 4 weeks in advance. A half day, a single individual day or up to five consecutive days can be booked at any one time.  Costs for use are listed below. You cannot book two sets of five consecutive days in consecutive weeks, without prior agreement with the EPPF staff. 


Click here for Auto-iTC200 online booking


24 hours’ notice should be given if a booked slot cannot be used.  If the machine is booked and not used, the booked user will be charged a nominal fee (click here for details).  The system administrators can delete booked user slots with 24 hours’ notice.


Please note that if you are using the machine and it is damaged or misused the PI responsible for the user is liable for the cost of repairs.  If you don’t know what you are doing, do not attempt to use the machine, please speak to Martin WearLiz Blackburn, Matt Nowicki, or Sandra Bruce.

 

contacts:


Facilities Manager:

Dr. Martin Wear

Rm. Swann 3.20

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: martin.wear@ed.ac.uk


Lab Manager:

Mrs. Sandra Bruce

Rm. Swann 3.19

Tel (+44) 0131 6507054

Fax (+44) 0131 6507055

email: sabruce@staffmail.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

MicroCal Auto-iTC200