Regarding the ITC question:  The relative sizes of the two proteins shouldn't matter when you're deciding which to put in the cell vs. syringe.  In fact, many people will run ITC expts. with Protein #1 in the cell, and Protein #2 in the syringe, and then also repeat the experiments, now with Protein #2 in the cell, and Protein #1 in the syringe.  If you get the same physical values (thermodynamic parameters and Kd, for instance) with both configurations, that's a good sign.  The main practical consideration here, is whether a particular protein can be sufficiently concentrated to go in the syringe.  If one of your proteins is more 'problematic' with regards to getting it up to high concentration, then maybe you will have to run the experiment with that particular protein in the cell (at lower concentration) and the other one will have to be in the syringe.  It's better if you can run the experiment with both configurations, but plenty of papers have been published with ITC data in which only one configuration of proteins in cell vs. syringe was used.  

The problem of using larger protein in the syringe is that, it will be at very high concentration in terms of mg/ml, and thus it might oligomerize/aggregate.

Urea is a widely used denaturant. In ITC, you must make sure the buffer condition in the syringe and cell are exactly the same. Never never inject denatured protein (say in Urea buffer) into protein in normal buffer, since heat release from dilution of urea will dominate.

In regards to your last question, do you mean purifying the protein in denaturing conditions? If that's the case, you could use both guanidine or urea. I'd recommend urea since you cannot run SDS/PAGE on samples in guanidine.

There are different ways to refold your protein: gentle dialysis, refolding in affinity columns or straight in a gel filtration column. The latter can be dangerous since some precipitation could occur in the column.

Jessica: 

Thanks for your answer! Yes, i have a result that can fit with multiple site model and I am going to switch the proteins from syringe to cell vice versa. I am wondering should i also make the concn different same as the previous experiment? For example, in the previous experiment, protein 1 (syringe) was 1mM and titrated to protein 2 with 1uM. If i switch the position of the two proteins, the conc of protein 2(now in syringe) should be 1mM and protein 1( now in cell) to be 1uM??

Jiahui Tao:

Thanks for your reply! Yes, i am using the same solvent for both proteins in cell and syringe. I have tried different conc of urea: 8m, 4M and 2M. I have run the urea-rea runs, the ITC graph showed endothermic peaks increased gradually for the last few injections for 4 and 8M while 2M urea showed

The syringe always has 10-100 fold more concentrated protein than the cell. In your previous dataset, what is the volume of sample in the cell and what is the volume of each injection? 1 mM Vs. 1 uM sounds not right. Suppose your cell volume is 1 ml, each injection is 3 ul, you will saturate the binding on your first injection. 

Based on http://www.endocytosis.org/techniqs/ITC.html, protein concentration in the cell should be around 30xKd. I suggest you start with 30 uM in the cell and 600 uM in the injector.

As for the peaks show up in the late stage of the experiment, I guess it is caused by injecting bubbles into the cell and make the noise. Just personal views.