In Isothermal Titration Calorimetry (ITC) heat changes associated with chemical reactions are monitored. It is a powerful, label-free, direct method to observe the thermodynamics of binding reactions and to directly characterize enzyme kinetics.
The binding constant (Ka), the stoichiometry (n),听螖H, 螖骋,听补苍诲听螖厂听can be determined from a single binding experiment. 螖肠辫听can be determined from the temperature dependency of 螖H observed in multiple experiments at different temperatures.听ITC of enzyme-substrate reactions can be used to determine KM听and kcat.
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Exciting Possibilities in Biology, Biochemistry and Biophysics
Ka, 螖H,听螖骋,听螖厂,听螖肠辫 (protein-protein, protein-ligand, DNA/RNA-ligand, DNA/RNA-protein, protein-lipids, peptide-lipids...)
听Measure heat 听directly
Avoid coupled enzyme assay or standard curves
Avoid labeling听or听immobilization听
Use turbid or colored solutions
Determine multiple binding affinities and stoichiometries in one system
Users have to receive hands-on training from Dr.Erbse, before they are allowed to use the instruments independently. Interested users should contact Dr. Erbse to schedule training. The training will cover sample preparation, performing measurements, cleaning the ITC and data evaluation. Training will be done employing a standard sample (Ca +2 / EDTA titration and water into water) in addition to an actual sample provided by the user.
The protocols give step-by-step instructions for a basic ITC experiment assuming samples have been prepared correctly and the user has been trained on the instrument. Reading the protocols Does not replace the training provided by Dr.Erbse.
After your training is completed you will be invited to join the ITC Google Calendar. To reserve ITC time use the Google Calendar to 听sign up with your name and lab. Please be sure to avoid booking times that overlap with other users
VP-ITC is located on the third floor of 听JSCBB in the D-Wing, room D381, on East Campus. Proxcard access is required after normal hours and on weekends. The ITC 200听is 听located in the听 Batey groups laboratory (fourth floor of the JSCBB, A wing room A455). After hour and weekend access must be coordinated with the Batey lab.听
Regular user groups are expected to opt into the pool and pay an monthly share that goes towards the general up keep of the Shared Instruments Pool. For detailed information please contact Dr. Annette Erbse.
Users are expected to provide all consumables specific to their experiment.
Users are expected to pay for repairs or parts that are necessary due to damage caused by their carelessness or neglect . This includes replacing the titration syringe should it be broken or the needle bent.
Cost for repairs/parts/services resulting from 听normal wear will be split based 听on the time used between all user groups
Please Note -
Since repairs/service associated with normal wear might not be necessary every year, depending on use, the cost听might come quite some time after use.
We have a VP-ITC and an ITC200听instrument from MicroCal. Both instruments can directly measure听 affinities from the high micromolar to low nanomolar听 (KD颅 = 10-颅3 to 10-8 M).
The VP-ITC has an active cell volume ~ 1.4 ml, syringe volume 0.2 ml
the ITC200听has an active cell volume ~ 200 ul. syringe volume 40 ul
