Hello all,
I am working with a protein X for which we are running an ITC (Isothermal Titration Calorimetry) experiment with a ligand ATP. When we have performed the initial experimental standardizations, we realize that HEPES (pH=7.5) but not TrisCl is the suitable buffering reagent for our experiment. At that time, we did not face precipitation.
As our instrument is a pretty old model (which requires large quantities of the protein), we are now terribly facing the precipitation of the protein while dialyzing against the Buffer (HEPES pH=7.5 50 mM, 250 mM NaCl, 5% glycerol). We are using a 2 mg/mL concentration at this step.
We would be very thankful for the kind suggestions from anyone who had a similar kind of experience or/and faced this problem before. Suggestions are welcome for the ITC specifically, we don't have to switch over to other techniques.
A 1-unit change in pH and/or a substantial change in salt concentration (probably by removing it) would be my first thought.
Perhaps you can try buffer exchange columns. It is fast and your proteins may not precipitate. Do buffer exchange and concentration just before the experiment. Also, consider the pH adjustments.
Thank you,
Amal.
Dear Manoj,
Your protein can be more soluble in a mixture of water and DMSO (dimethyl sulfoxide). DMSO is used in the buffer to improve the solubility of hydrophobic proteins. Make sure you have the same final DMSO concentration in both solutions (protein and ATP). The recommended upper limit is 10% DMSO for ITC binding studies. In my experience, I had problems with the dimerization (and precipitation) of a protein, so I used a reducing agent (2-mercaptoethanol). Hope these procedures could help you.
Best,
Rogério.
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