I have a mixture of monomer and dimer. Monomer of the protein has N terminal His tag and i did not remove the his tag after purification. I provide particular condition to my monomers so that two monomers interact covalently to form dimers. But all the monomers will not form dimers some protein will be in monomeric form.
As dimers has more His so it will bind to column more tightly than monomer.
I would not recommend to use Ni-NTA column for separation of your His-tagged monomer from your dimer. There are too many variables that you can't predict, such as one of your His-tag might not be accessible in the dimer for the binding to the column, the conformation of the His-tag in the dimer might not be in the best position for highest affinity binding, and so on. I would suggest first to purify both monomer and dimer together on Ni-NTA column, and after that separate them by gel-filtration column.
Dear Sir,
Thanks for suggesion..i tried even gel filtration but didn't work.
https://www.researchgate.net/post/i_want_to_separate_protein_dimer_46KD_from_protein_monomer_23KD4
Also whta would be if i use denaturing conditions. Same as that of we generally use for purification of insoluble protein from cell pellet.
what is your opinion on ion exchange chromatography for purification
I think it would be interesting to try the Ni-NTA chromatography. If both tags are freely accessible in the dimer, the affinity of the dimer for the resin may be higher than the affinity of the monomer. You should use a shallow imidazole gradient elution to maximize the chance of benefiting from a small difference in affinity. If you try this experiment, please let us know how it worked out.
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