Hello everyone,
I am struggling with the purification of a recombinant protein (6xHis tagged). The overexpressed protein binds to Ni-NTA-agarose beads adequately. However, it is not eluting out after binding to IMAC resin. Several elution conditions, including different concentrations of imidazole (0.2 - 1 M), low pH buffer (pH 4 - 6), or 8 M urea, have been tested. Nothing has worked. Purification of the same protein with GST-tag has also been attempted using GSH-Agarose resin. There too, elution with reduced GSH is inappreciable. Tried to elute the protein by in-bead GST-tag cleavage applying TEV enzyme. But, post-cleavage protein remains bound to beads only and cannot be eluted. Any suggestion...why is this happening? How to improve the elution of protein?
There is no way that a polyhistidine tagged protein would not be eluted from a Ni-NTA resin with 1 molar imidazole or with pH 4 buffer. Either you are not able to recombinantly express your protein in cells or your Ni-NTA resin is totally stripped of nickel ions or your protein doesn't actually have a hexahistidine tag (Do you have the DNA sequencing results showing that?) I am betting that you are not expressing your protein. Do you have SDS PAGE gels of your lysate, supernatant, pellet, and flow-through from Ni-NTA?
Thank you, Nick Gee & Adron Ung for your prompt responses...The plasmid construct coding for two proteins: protein-A (6His-tagged at N-terminus; trying to purify) and protein-B, has been confirmed by DNA sequencing. The expression of recombinant protein (6His-A) has also been confirmed by immunoblotting using anti-6His antibody and protein-specific mice anti-sera (both in lysates as well as after binding to Ni-NTA beads).
For your reference, I am hereby attaching the gel images of protein purification samples. Hope this may help you in better interpretation.
These Coomassie stained SDS PAGE gels show that your protein is binding to the beads and coming off with 250 mM Imidazole. You might have bound so much that you need a larger volume of 250 mM elution buffer to get all the protein off the beads. You can just monitor the drops as they come off the column by the absorbance of those drops at 280 nanometers monitor the spike for a lot of protein coming off and watch it drop to zero.
Thank you, Adron Ung for your kind suggestion...Will try the same
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