My student is trying to set up an ITC experiment using Nano ITC low volume model. I get conflicting information from reading the company manual. It states the sample chamber volume is 190 ul, but somewhere else the manual states to use ~300 ul sample for an ITC experiment. Wouldn't it cause an overflow situation? In general the manual avoids give out specific numbers for setting up an experiment. Can anyone let me know their favorite numbers to use for this model type?
Hi Ailong,
I don't know exactly the context of the two volumes mentioned in the manual. I guess the higher volume is recommended for a bubble-free transfer of your sample from the reaction tube to the sample chamber. Based on my experience I can say that you should prepare 300 ul of the sample for the experiment, as this prevents the incorporation of bubbles while filling the loading syringe (when you collect the sample from your reaction tube) and also the sample chamber. You need not use up the full 300 ul of the sample but make sure that you fill the sample cell slightly on the higher side in order to avoid generation of bubbles during the experiment as well.
Hi Ailong,
I don't know exactly the context of the two volumes mentioned in the manual. I guess the higher volume is recommended for a bubble-free transfer of your sample from the reaction tube to the sample chamber. Based on my experience I can say that you should prepare 300 ul of the sample for the experiment, as this prevents the incorporation of bubbles while filling the loading syringe (when you collect the sample from your reaction tube) and also the sample chamber. You need not use up the full 300 ul of the sample but make sure that you fill the sample cell slightly on the higher side in order to avoid generation of bubbles during the experiment as well.
Hi Ailong,
Most commercialized ITC cell volume are 190 or 200 uL for low volume version. The cell volume mentioned here is “active volume” which means real volume involved the reaction. In other words, you need prepare more sample to load into sample cell to meet active volume. The suppliers want everyone to get good data so that give a safe number for instance over 300 uL. In addition, the signal might not easy to interference by room temperature if you loading more sample.
You might doubt why 300 uL why not 250 uL? I guess maybe related to the cell shape, stirring needle, and paddle design. The factories already test and gave user the recommend number. I remember the suggest volume is over 300 uL.
To follow up on these two comments: (1) To avoid bubbles, you must degas the samples for both cell and syringe beforehand; a degasser was probably purchased with the instrument. (2) As noted by Lin, most instruments these days are of the perfusion design, meaning the cell is overfilled from the start and is progressively more so after each injection. The active volume ends at a narrow fill tube at the top, which is probably a couple of cm in length, opening into a wider overflow region above that. For the Malvern instruments, the filling procedure described in the manual is designed to fill to just the top of the fill tube, leaving the entire overflow region to accommodate the material expelled from the active region with each injection. This does not require premeasuring the volume of the cell reagent. However, one could always start with a measured amount large enough to ensure that the level is a bit above the top of the fill tube when the syringe is installed. Perhaps that is the reason for the 300 uL, but I would think this value does not need to be followed precisely, i.e. 10-20 uL more or less is probably O.K.
Does anybody know the exact active volume of the measurement cell of the NanoITC low volume device? Is it really 190 uL, or is it 170 uL (because the volume occupied by the syringe has also to be considered) -- which volume do I have to use for the calculation of the binding parameters?
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