Ni-NTA purification of His-tagged fusion proteins

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MelanieBiotech
MelanieBiotech's picture
Ni-NTA purification of His-tagged fusion proteins

 Hey,

I would be really greatful if anyone could give me some hints regarding my problem..

I am currently trying to purify fusion-proteins with a His-tag which are not expressed really good, but the even bigger problem is, that after Ni-NTA purification and Western Blot I find that my proteins are always in the flow through, even though His-tag (sequence) seems to be fine... proteins are expressed in HEK cells and secreted into the medium. I always adjust the pH to 8.0 and perform binding in batch for 1 h.
What is really weird is that one time, there was protein in the elution fraction as well as in the flow through BUT the band in the elution fraction was of lower molecular weight (about 10 kDa). The His-tag is at the C-terminus... if something was cut off, it can't be on that side, so I don't understand why somthing with lower molecular weight should suddenly bind?!

I don't really know how to interpret all this and would be grateful for any help and advice!!

Kind regards,
Melanie

Chin Fen Teo
Chin Fen Teo's picture
Hi Melanie,

Hi Melanie,

Can you please tell us what are the components in your lysis , binding and wash buffer?

Thanks.