I did ITC experiments with BSA and Its difficult to get same results with same concentration of protein as well as binding ligand.
The first thing to do is clean the cell. This is absolutely crucial for getting reproducible results.
Thankyou Adam for your reply. Yeah I always used to clean sample cell with aq. solution of contrad at temperature 75C. Still facing this issues with results reproducibility.
- Test performance of instrument with reference reaction (e.g. Ca-EDTA titration)
- Test quality and reproducibility of sample preparation (purity, homogeneity, structural integrity, stability... regarding time, pH, ionic strength...) using standard biophysical techniques (SDS-PAGE, gel filtration, DLS, DSF, CD, DSC...)
Each of these two points may involve a considerable work, but it will save time and misguided troubleshooting, and, these tests should be performed anyway
Thankyou for your reply!!! I used to do UV, DLS and pH analysis before ITC experiments considering concentration &pH as main parameter. Shall I need to perform all these before ITC. One more thing my binding ligand is of different pH as compared to protein solution in sample cell. will this hamper the results anyway?
Once your sample passes those test systemstically, you may reduce the number of test.
Both solutions, cell and syringe, must be as identical as possible (buffer, pH, ionic strength, co-solutes...). If pH in cell and syringe is different, the heat effect from neutralization after each injection may be even larger than that of the interaction you want to detect and measure, and a large background heat will arise.
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