The protein I purified is in Tris buffer sitting at -20. I want to do ITC assay for protein-ligand binding studies. I am planning to do buffer exchange with phosphate and citrate buffers at pH 7.4 and see if my protein is happy with it. Can anyone suggest what other components i need to consider, like glycerol, Nacl? i wish to use BME as TCEP is little expensive.
Any other suggestions regarding the ITC buffers are welcome as am new to these assays ..
for ITC reactions, you can use any buffer that is suitable for your protein to keep it soluble through out the reaction. You should consider having a buffering medium (Tris, or Phosphate buffer), NaCl, any co-factor your ligand requires for interaction with your protein (eg. Mg+2 or Mn+2 is generally required for ATP) All other components depend on the the solubility of your protein. Avoid having too much reducing agents as they may harm ITC cell.
I did my ITC experiments with 20mM Tris pH 8.0 and 150 mM NaCl. 5 mM MgCl2 was added with ligand (ATP, ADP etc) solution.
First of all do not use citrate buffer. Phosphate buffer at pH 7.4 is OK. Check the co-factors needed for your protein to show optimum activity. Finally avoid trying to replace TCEP. ITC buffers work well provided you attend to minor details like ligand concentrations etc.
I suggest you try a buffer that is compatible with your protein. This is, that it will not precipitate the test protein at the concentration in the cell. Also, it should be compatible with the ligand. Said that, use the buffer for the last dialysis of the protein to dissolve the ligand. This helps to make the blank measurements to determine the dilution heat.
Besides the suggestions gave by the colleagues above, it is worth to mention the ionization enthalpy of the buffering system as another important property to take into account. If you want to discard possible protonation/deprotonation events linked to the binding process you must use different buffer systems at the same pH but with disimilar ionization enthalpies (v.gr. at pH 7.4 you can use phosphate buffer, DHioniz= 5.1 kJ/mol, or triethanolamine buffer, DHioniz= 33.6 kJ/mol). It is also important to maintain controlled the ionic strength of the buffer, usually at 0.10 or 0.15 M, adjusting it with a "neutral" salt within the Hofmeister series, like NaCl.
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