I am using Ni-NTA method for protein purification. Due to buffer constraints I want to elute the protein with Histidine. Can anyone let me know what conc. of His I should include in my wash and elution buffers?
Hi there,
I guess the range of concentrations should be the same as when using imidazole: up to 50mM for washing and up to 0.5M for elution. BTW what is actually the advantage of using histidine over imidazole?
Hi,
I have purified many His- tagged proteins and, according to my experience, histidine is a stronger exchanger in comparison to imidazole. I generally work with proteins which have a 6-Histag at the N-terminus and what I do is to perform a first wash with the protein buffer containing 1 mM His, than I increase the His concentration to 5-10 mM and finally I elute the protein with 40 mM His. Alternatevely, what you can do is to perform a gradient from 1 to 40 mM collecting small fractions and see what is the concentration at which your protein was eluted.
Good luck!
Hello Dominique and Valentina,
Thank you very much for valuable inputs. I will try to titrate histidine conc. Imidazole is not compatible with my assay but histidine is. How to you prepare the histidine solution? I tried to make 1M histidine in tris buffer pH7.5 but it does not seem to dissolve.
Check the maximum solubility of histidine in water solutions. Probably 1M is too much. I generally prepare a stock of maximum 100mM.
Hi again,
According to SigmaAldrich, limit of solubility in water is 0.25M (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Product_Information_Sheet/h8000pis.pdf).
The following paper describes elution with gradient of histidine up to 0.25M : http://www.sciencedirect.com/science/article/pii/S1366212008700989
@ downvoter : what's wrong in my previous post? I've only mentioned data from a chemical company and from a published paper. What's wrong with these then??? Thanks for having the gut to give the explanation...
- Badges
- Science method