The two materials are very complicated polysaccharide and proteins. So, vibration is somehow expected. However, after each exothermic reaction, an endothermic one is occurring; which I do not have any idea about. Moreover, the baseline is shifting down. I am wondering if I am allowed to normalized the baseline after subtracting the control data? if yes, how?
It seems to me that you have a few kinks to work out in your ITC to get a good curve. There are many explanations for why you have both an exothermic and an endothermic peak for each injection, and you will need to go through a number of controls to verify whether that is inherent to the binding reaction or a mixing issue (buffer issue) or something else..
Regarding the base line it is obviously drifting a bit, but that's in itself not a big problem. As the baseline is calculated throughout the curve, the integrated peaks will be fairly ok anyway, at least after you have manually corrected the baseline in the software (Origin, if you use a ITC200). So no need to normalize it, simply subtract your control data.
I'm unfamiliar with correcting baselines in Origin, but you could try NanoAnalyze (TA Instruments). It's free, easy to use and automatically adjusts the baseline from your raw data. NanoAnalyze can also normalize the baseline in the Overlay tab.
Like Ronnie mentioned, go through the controls first (buffer-buffer, polysaccharide-buffer and buffer-protein) and repeat the experiment again. I'd also recommend reviewing the literture how others setup up ITC experiments including polysaccharides. You'll never get the perfect isotherm on your first try!
I agree with what Ronnie said above. I just want to point out explicitly that you always have multiple reactions occurring when you do this sort of injection calorimetry. We often ignore a couple of the reactions because the amount of heat is very small, but that may not be the case with you. Remember that at a minimum you have:
1: polysaccharide-protein association
2: polysaccharide dilution as it expands into the larger volume
3: protein dilution as it expands into the larger volume
Any one of those three could be endothermic, with the second one being a real possibility. You also could have
4: higher order polysaccharide-polysaccharide association or dissociation
5: protein-protein subunit dissociation consequent to polysaccharide association
Either of those latter two could result in an endothermic reaction. If you have access to analytical ultracentrifugation, you can test whether the latter two possibilities are true. I would first do the blanks Ronnie, recommended, though. Those blanks will establish whether the dilutions of #2 or #3 are responsible for the endothermic reaction.
Oh... and I forgot the obvious one, that would also be found from your blanks:
6: dilution of buffer components and salt background components. What's in your buffer? Does the buffer background of your polysaccharide and protein have an exact match? Co-dialysis may help, if you don't have an exact match.
Dear Ronnie, Nicholas and Benjamin,
Thank you for your time and help. I am doing the buffer analyses and subtracting as Ronnie said. Moreover, I redid the test with lower concentration and this time I got a normal ITC curve at first 10 injections (exothermic) and from 11 to 20 injections were endothermic (???). I will be back to you again when I am done with a few more tests and buffer subtraction.
Thank you again.
Saman.
You need to be sure that your actual DP value (the Y axis of the raw data) is always above zero ucal/sec. Your data are all below zero. What is your reference power setting? The baseline value needs to be within 1 ucal/sec of your reference power setting. The reason for your original data in the PDF is showing both exo- and endothermic peaks is that the MicroCal ITC cell heaters are overcompensating for the negative DP value. Make sure your reference cell is filled, and your ITC cell is clean and there are no bubbles in the sample cell.
I got the same problm before. simply decrease the conc. of ligand in syringe. you will get one type of graph.
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