I'm trying to purify c-terminus his-tagged membrane protein that is stably expressed in HEK293S cells.
I extracted membrane protein using detergent and confirmed the presence of the target protein using western blot.
But after Ni-NTA purification, there are a lot of nonspecific band by CBB stain.
I washed the Ni-NTA resin with phosphate buffer containing 150 mM NaCl and 40 mM imidazole and eluted the protein with same buffer containing 500 mM imidazole.
Target protein was detected by anti his-tag antibody.
How can I improve the purity of the protein?
the problem maybe occur after purifing.
i don't know whether you heat the samples before Western blot. try to load the samples immediately after adding the loading buffer(or warm at 37 degree for 5 min) .
Presumably you also had detergent in your wash and elution buffers? I would try a gradient of imidazole rather than a large step to see if the contaminants and your protein elute at different concentrations.
I've heard that His-tag is not ideal for purification of proteins expressed in mammalian cells because the cells contain other sequences that bind to the resin. Try another purification tag or add another purification step, such as ion exchange chromatography.
Thank you all.
First I forgot to write about detergent.
I added detergent in all buffers used in this experiment.
I read several papers that use HA tag or FLAG tag for mammalian protein but the resins are very expensive so difficult for us.
I think imidazole gradient and ion exchange is good idea I'll try them.
I still need more idea.
You did not say how long your his tag is. We have used 6 his tag and 12 his tag and often find a longer tag is better.
To quote the Qiagen Expressionist manual:
"Purification of 6xHis-tagged proteins expressed in mammalian cells: Purification of 6xHis-tagged recombinant proteins expressed intracellularly can pose
problems"
First of all I would strongly recommend against using a 6xHIS tag in HEK cells, try using FLAG. In case you can't change the tag anymore, try reducing the amount of NiNTA resin. Hopefully your protein will compete with the endogenous contaminants for less binding sites and your HIS-tag should have a higher affinity. Also, try to increase your protein concentration by using ultracentrifugation and then solubilize your membranes. There should be less contaminants after this step.
Good luck!
I would agree with Maximilian about reducing the amount of NiNTA; this should hopefully reduce some non-specific binding of proteins to the resin. Another thing you could try in parallel is increasing the concentration of imidazole; you could try using: up to 20 mM in the binding buffer, and up to 60 mM in the washes (I get very good purity for my his-tagged proteins under these conditions). This should further decrease contaminants.
I would also look at the detergent you are using a good starting point is DDM or LMNG or if your protein is super unstable something like digitonin. If you are suffering problems with purity we have successfully used a double STREPII tag which tend to pull down very few non specific contaminants
good luck
Thank you all.
I'm using his6 tag and detergent is DDM.
I've also found that his6 tag is not good for mammaｌian cells especially to purify cytosolic protein.
So I'm wondering soluble faction completely removed membrane protein is also difficult to purify.
And I also found several reports that mention the success of his-tagged protein using TALON cobalt ion chelated IMAC.
I'll also try using less amount of Ni-NTA.
to James C Errey.
Thank you for your suggestion using STREP tag.
My purpose of the study is crystal structure so I hesitate to use longer tag and I don' want to remove the tag because it makes the experiment complicated.
I want to use single STREP tag, is it enough to purify the protein?
Still need more suggestion.
Best
Kiyohiro Takahasi
Hi , Kiyohiro,
Have you solved your problem? I met the same promblem as you put up with here.
I added 20 mM imidazole in the binding buffer, and 80 mM in the washes, 500 mM in the Elution. There were a lot of nonspecific band by CBB stain. And my protein was not the strongest one.
Should I have to change the tag? I have another tag Myc between my protein and His tag.
Any advice?
Thanks ,
Jianying
Hi, Shen
Unfortunately, I could not find a way to purify his-tagged membrane protein from marmarian cells.
Purification protocol is same as your method and result is also same.
Now I'm trying 1D4 tag.
http://www.cube-biotech.com/background-tips-and-tricks/what-is-rho-1d4
From several reports I read, purification with antibody is the best way.
As long as I know, reports using 1D4 tag and Flag tag showed the good purification results.
I think that you might want to try anti-Myc tag antibody conjugated sepharose.
Best
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