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ridhima

elution buffer pH

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My question is that when eluting protein with elution buffer we set pH of elution buffer (sodium phosphate buffer) to be 7.5 but after adding 500mM imidazole pH of buffer increases to 9. I want to know is there need to adjust pH of the elution buffer after adding imidazole to 7.5. If yes, then how can we set the pH by using NaOH or HCl or by adjusting with sodium monobasic or dibasic buffer salts or make buffer of less pH like pH 6.0 and after adding imidazole the pH will reach 7.5 after doing the standardizations that how much pH buffer to make so that after addition of imidazole it reaches 7.5 pH.

 

 

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The pKa for the imidazole conjugate acid is of around 7. You do not want your pH to go any lower than that, as the imidazole will become protonated and won't bind to the resin (might still get some elution from protonating the his-tag, but it's not very efficient). At pH 9, the imidazole will not be protonated, so it will still elute your protein. However, your protein will remain in the elution buffer, and for many proteins a pH of 9 is not optimal.

 

I would add HCl until the pH goes down to 7.5. Add a bit, mix, measure pH. Repeat until you get to 7.5.

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