Hey, I need help! I'm trying to purify one proteins (82 kDa) using a Ni-NTA resin. The enzyme have a 6-histidine tag (bioinformatics models show that the tags are exposed correctly), and expression occurs in E. coli (strain BL21). The problem I'm having is non-specific binding, the elution comes out as dirty as the original sample or flow-through.
I have already performed the purification on HPLC and on a bench column, by gravity, but both show the same result in the gel: several impurities. I have also varied the buffer pH and composition (I used CHES pH 9.0 and HEPES pH 7.5). Furthermore, as I saw that many researchers suggest adding imidazole to the binding buffer, I added 10 mM of imidazole, but this was enough for my protein not to bind to the resin, but all the other contaminants did. I wonder: what is there in the sample that has such a strong interaction as a his tag?
Oh, I have also changed the bacterial strain and the pattern remains the same. These results are from yesterday: I resuspended in 15 mL of buffer the equivalent of 750 mL of expression medium, supplemented with protease inhibitor. I centrifuged, filtered and eluted with a flow rate of 1 mL/min. I have a nice elution peak, but when I load the collected fractions into an SDS page. There are so many proteins, and they all elute together in a single peak! (Sorry for the picture of the SDS page, I took it with my phone. The collected fractions are the last five. The bands outlined in green refer to the peak.).
It is worth mentioning that I am working with proteins from the synthetase class, three proteins to be exact, and they all show the same behavior, with the same peak in the same place. However, we purified a GAPDH without any problems. Could it be something from the class?
It seems to me that you have a problem with the binding of your protein to your column (on SDS-PAGE: presence in the flow through and the rinse?)
You can try this protocol :
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011700_HisPur_NiNTA_Resin_UG.pdf
Buffer A : 20 mM sodium phosphate, 300 mM sodium chloride pH 7.4
Buffer B : 20 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole pH 7.4
In second step, If not clean add 10 mM imidazole in buffer A
Thanks! and no, it's not coming out in the wash... it's binding to the resin, I think. sorry for asking, but how can changing from HEPES to phosphate improve purification? oh, my protein's pH is 8.2, so I use pH 7.0
It is instructions for use column and recommended buffers, here :
https://cdn.cytivalifesciences.com/api/public/content/digi-13164-pdf
You choose a buffer that allows you to fix well, to keep your protein functional and to limit contaminants. CHES pH 9.0 and HEPES pH 7.5 doesn't seem to give you good results.
yes you can try pH 7.0 or 7.2 if isoelectric point of your protein is 8.2 it will be better to do it like this
Steric hindrance can be a cause making histag inaccessible...though you checked it with modelled structures, we don't know it for sure. U can verify it by trying to purify the protein under denaturing conditions by using urea or GnHCl.
Do you think that because the protein is in the soluble fraction the urea concentration can be less than 8M? Since 8M is generally used to remove from inclusion bodies.
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