Hi.
When I purify my His-tagged membrane protein with IMAC, I label the protein (containing a single cysteine) with fluorescein-5-maleimide while the protein is immobilized to the Ni2+ NTA. I wash extensively to get rid of impurities and free dye, however, while this procedure has worked perfectly half a year ago, now it seems like the dye is somehow interacting with the resin. Consequently, what appears to be free dye is not washed away easily and ends up in the eluted fractions. My question is whether any of you have any idea why this is suddenly happening. pH, temperature, incubation time, molar stoichiometri, protein purity are all as they should be. Further, reagents are freshly made.
Thank you very much in advance.
I sadly cannot say where your problem originates from. But it seems that you have to handle impurities now that were not present in before right?
I personally think that this is because you use a His Tag and Ni-NTA to begin with. Maybe you changed from one resin batch to another? Because Ni-NTA based His Tag Purifications are meant for high yield purifications with a tendency for impurities.
Since membrane proteins do not have the greatest abundance even under a strong promotor you should change to an affinity chromatography method that is more on the balance pf purity than yield.
For membrane proteins I would recommend the Rho1D4 Tag on the proteins C-Terminus: https://cube-biotech.com/what-is-rho1d4-an-affinity-tag-specialized-for-membrane-proteins
But if you want to continue with the His Tag, I would at least change to Co-NTA instead of Ni-NTA. Co-NTA may have less protein yield than Ni-NTA but the purifications are much cleaner.
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/co-nta/ (The Page includes a small diagram that compared ions in terms of purity and protein yield for his tag purifications)
Hi Sebastian.
Thank you for your answer. I ran an SDS-gel of my recent purification against protein from a previous batch that worked perfectly. The purity looks to be the same by eye. However, the assays that we are performing are extremely sensitive to even homeopathic amounts of free or unspecifically bound dye, so I might definitely tro Co-NTA resin! Thank you kindly.
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