I can't understand that how PH cause his tag proteins bind to Ni-agarose resin or come off it. I want to know more about interaction. can anybody suggest a good article about that? for example in some literature decreasing PH helps remove contaminant in elution, but how?
Hi Fereshteh, histidine is a unique amino acid that binds metal ions and this binding is dependent on whether it is protonated or deprotonated. Above pH 7 it is deprotonated and binds metals. Below pH 7 it becomes protonated and does not bind metals.
Hi Fereshteh, histidine is a unique amino acid that binds metal ions and this binding is dependent on whether it is protonated or deprotonated. Above pH 7 it is deprotonated and binds metals. Below pH 7 it becomes protonated and does not bind metals.
Any interaction between biomolecules is to a certain extent dependent on the buffer conditions, especially ionic strength and pH. I recently saw a very nice example for a protein-protein interaction with highest affinity at pH7 and a salt concentration of 150mM NaCl. Changing the conditions led to a decrease in affinity.
His-tagged proteins bind best to Ni-agarose resin if you are using a slightly basic pH (7.5-8). If the pH gets more acidic, the Histidine residues will be protonated and thus are not binding to Ni2+ resin anymore. You can also check the website of the Ni-resin manufacturer to get more information on that.
Hi Mangal, sorry for the nitpicking, but the histidine imidazole function that is responsible for the coordination with metals is not negatively charged above its pKa (~6-7). It is either protonated or not, depending on the pH.
His possesses an unbonded electron pair on N, to which Ni2+ wants to bind as it say lacks two electrons being two times positive. It is not an electrostatic type of interaction, it is called coordination bond. H+, proton, competes also for this electron pair of His. I would add also that HIS is not unique amino acid that binds metal ions, as there is a whole science called Bioinorganic chemistry, which investigates binding of metals to proteins and DNA, RNA etc. Transition metals bind to His, Cys, Met, Arg, Lys, anywhere where there is a free electron pair, say on N, O, S....I would say that the His - Ni2+ combination was chosen because these two form the strongest bond.
Hi Romana, I should have clarified that HIS is a unique amino acid in that that it can coordinate metal ions and release them within a physiological pH range.
From this question, I would like to know between pH2-7, the effect of binding is it same or not? Because I am not sure what factor that affect to protonate such as pH2 will protonate more than pH7 and it means HIS still can bind with Ni in pH7>pH6>5>4>3>2... like this or it cannot binding since pH lower than 6-7
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