Section 6: Tips and Troubleshooting
Bent injection syringe needle
– Remove the syringe from the pipette and roll on flat tabletop.
Watch the needle to see if there is any wobble, indicating it is not straight. A machinist using a
lathe can sometimes straighten bent needles.
Syringe height adjustment –
The syringe height adjustment (distance from the bottom of the cell
to the bottom of the syringe stirring paddle) is important for optimizing the performance of your
ITC. This distance has been found to be approximately 3 mm. Once this adjustment has been set
it is usually good for the life of the syringe, but new syringes must be set according to section
PROBLEM:
The baseline seems to drift over time and appears cyclic.
SOLUTION:
Check to see if the ITC cell is in the direct path of a heating/cooling vent. If so, relocate the ITC
away from air currents or direct the air currents away from the ITC. Also, the baseline may drift
more than desired if the room in which it sits is not stable in temperature. Try to provide the ITC
with a well thermostatted environment.
PROBLEM: When attempting to remove a pipette plunger tip, the tip removal tool is
unable to grab the tip, with the tool slipping off the tip instead.
SOLUTION:
To remedy this you must move the pipette plunger down slightly. One can use the Dn (down)
Distance button in the Pipette Controls group of the ITC Controls window to move the plunger
ca. 0.1” - 0.2” downwards. The tip removal tool will then be able grab the plunger tip.
6.3 A Note About Buffers
DTT (Dithiothreitol)
– If possible, DTT should not be used in the solution for ITC experiments.
Since some proteins are unstable without DTT, the presence of DTT in solution should be as low
as possible. Experimental data suggests that even 1 mM DTT could cause a large artifact in the
ITC baselines. One way to minimize the artifacts is to remove oxygen from the solution by
purging with argon (better) or nitrogen.
TCEP ((tris(2-carboxyl)phosphine)
– Experimental data suggests that TCEP at low concentration
(i.e. 1 to 2 mM) have little effect on the ITC baselines, although at a higher concentration (i.e. 10
mM) it causes a large slope in the ITC baseline. For some proteins, it may be a good idea to
replace DTT with TCEP, but the user is cautioned to be aware of potential problems.
6.4 Selecting the Proper Stirring Rate
There are 5 stirring rates available, they are approximately 0, 270, 310, 500 and 580 RPM
(revolutions per minute). The exact stir rate for your instrument is measured and the value is
entered into the software. In selecting the optimum rate, there is a trade-off between mixing
efficiency and baseline noise. For almost all your studies, stirring rates of ca. 270-310 will give
adequate mixing following injections and still provide very high baseline quality. However, for
binding studies where the c value (i.e., the product of the binding constant times the concentration
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