Hello everyone,
I try to purify a chitin binding domain in a native conformation in mono disperse suspension. After purification by use of a polyhistidine tag I dialyze the protein suspension in PBS buffer. After the dialysis process I get precipitation and the protein doesn't bind well to chitin beads. Can anyone give me an advice why the domain doesn't bind to chitin after a dialysis process in PBS? Could the ionic strength be too high? In potassium-phosphate buffer the binding properties work well, but the suspension isn't mono disperse (DLS measurements)
Thanks in advance,
The binding to chitin binding domain to his-tag is affinity associated but definitely affected by high ionic strength of the buffer and or pH. Acidic pH of buffer shall discourage binding. So try to address these issues!
Dear Marco,
Are you performing a dialysis to facilitate buffer exchange? And yeah, as you assume and as Prof. Shamsher says, the high ionic strength could be a reason for a decrease in the binding. Another reason for the precipitation could be that your protein concentration is too high and they start agglomerating; higher the protein concentration, higher the interaction between the protein groups in the sample, thereby enhancing the protein:protein interactions. When such a protein sample exists in an aqueous solution, they tend to stay close together avoiding major interactions with the surrounding medium.
Thanks a lot for your advices. The PBS buffer has a molarity of around 150 mM and a pH of 7.3. Could this ionic strength be too much?
Would you either change the molarity of the PBS buffer or change the whole buffer (potassium phosphate, MES, HEPES, MOPS) and the molarity to 50 mM?
Dear Marco,
Just as Prof. Shamsher says, you may reduce the strength of your buffer. Else, a trial-and-error experiment with varying buffer strengths could help you decide the optimum. As an additional information that I'd like to know, is your protein concentration high?
As an update, I'd like to report, that the precipitation was a result of the reaction of CaCl2 and Phosphates. These crystallize during the dialysis process and minimize the protein concentration. Further the high concentration of PBS results in a decrease of affinity. Lower ionic strength results in a higher activity.
Thank you for the help to get this solution :)
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