Problem in purifying fusion protein with His Tag - Fusion protein found in Flow Through (Sep/26/2007 )

I had cloned my gene in a N terminal pQE expressing plasmid. Apparently when I purified my protein under denaturing condition, the fusion protein was found in the flowthrough during washing.

I had checked my sequence and I am sure it is in frame with HisTag as the fusion.

I had tried like few times and finally confirmed it was really found in the flowthrough.

Do you guys think it might be the washing buffer problem? or the protein itself?

Any feedback i is much appreciated.
Cheers

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is the protein soluble in the denatured form ?
did you good equilibrate the column (i had such kind of pb xith NiNTA agarose)
do you use enough matrix ?
you may try dialysis against binding buffer to help.
you may also use centricon to concentrate your protein.

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Thanks fred,

Yes, the protein is soluble in denatured form.
And I did equilibrate the column prior using.
I am using the spin column with the matrix embedded as well.

I am still thinking whether it is the pH problem from my washing buffer. And I do not have imidazole in all the buffers.
Argh... I still need to purify it.

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It has been so long since the last time I worked with His-tagged, and I don't use them much either, so don't know if I remember correctly, but aren't the washing buffer and the elution buffer quite similar to each other, only different in the amount of immidazole or something like that, right? So maybe the binding of your protein to the beads is weak, so it get eluted out just by using washing buffer? Maybe consider using that washing buffer as elution buffer instead and make something milder for the washing?

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How do you know that it is in the flow through? do you detect it with a specific AB against your protein or against HIS?
How long ist the linker between protein and HIS tag? Is it possible that there is a cleavage site inbetween? How do you perpare the protein solution which is loaded on the column? (cleavage while sample perparation)
How do you wash? HIS tags often have different affinities to the column depending on the fused protein (Which column by the way and which volumne?) so that it is possible that even low conecentartions of imidazol (like 20 mM) are sufficient to elute them.
If you elute by pH have you double checked your pH and also have you checked other pH values. Elution pH can also vary from construct to construct.

In general for HIS-purification use as less column volume as possible to prevent unspecific binding of other HIS moieties.

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Thanks for all the replies. I wasnt around. Went to Indonesia for few days.

Anyhow, imidazole is usually for native protein isnt it? My buffer is without imidazole. So shouldnt be that problem.

How do I know about protein in the flowthrough? I collected my flowthrough during purification steps and run under SDS PAGE. My boss was suggesting, maybe there are some LITTLE amount of protein stucked in the matrix. But I got a pretty decent amount of protein in my flowthrough and none (i mean really nothing) for elution.

I am using spin column with matrix being packed.
I am not too sure about the cleavage though. How do I check that?

ANymore advices are much appreciated. Thanks alot

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i facing the same problem few months ago.

i think the pH of ur lysis buffer, washing buffer and the elution buffer are very important. so check the pH eveytime before u perfom the assay to ensure the buffers are in the correct pH condition. i normally use freshly prepared buffer.

besides, i kept my buffer in cold, since it might enhance the binding between the column and the protein.

even though i follow the denaturing protocol, but i had add imidazole in my binding and washing buffer, this had improve the purity of my purified protein (recommended by qiagan).

wish u good luck.

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