Dear TriNgo,

Firstly, the fact that you can see your His-tag via Western blot does not
mean that it's attached to the majority of the protein molecules in
solution - strong Western signals may be observed for as little as 1% of
the protein that is *supposed* to be His-tagged. So one of the causes may
be that your protein is chewing itself up or is being chewed up by
something that causes most of the molecules to lose the tag (but for a few
percent to remain tagged and thus give you a Western signal).

The other alternative is that your protein is misfolded and/or aggregated.
I've seen several cases of aggregation resulting in His-tag being 'hidden'
away from the resin. The way to check this is to take your lysate and add
guanidine to 6-8M, then re-run the Ni-NTA column. If the protein suddenly
binds - then you know what's up.

Finally, your protein may be folded, but the tag may be 'concealed' in a
suitable protein pocket and thus isn't exposed enough to the solvent and
therefore to the resin. Common cases of such behavior typically involve
oligomeric proteins. Again, denaturation may suggest an answer,
alternatively just moving the tag may be the way to go.

Note that IMAC works better at pH 8-8.5 so if you want, you could repeat
your extraction at higher pH.

Finally, it is not common to see insect cell media interfering with the
binding of proteins to IMAC. If you have large amounts of medium in your
cell paste, you may want to either wash the cells (gently!) during the
initial pelleting process, or try adding ~5 mM MgCl2 which has been known
to help (presumably by complexing away the chelators naturally present in
insect cell medium). This may or may not work. You may also be able to do
primary separation of some sort (such as ammonium sulfate or PEG
precipitation, or perhaps bulk ion exchange etc.) prior to your IMAC step.

Best regards,

Artem

> Dear CCP4 users,
>
> I'm purifying a kind of protease having His-tag. The protein is expressed
> in
> insect cells and broken by sonication.
> I used NTA resin to purify this protein.
> Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl. Buffer B is 50mM
> phosphate buffer pH 7.5, 300 mM NaCl and 300 mM Imidazole.
> However, all proteins cannot bind to NTA resin. My protein is eluted in
> Flow-through. I also check the NTA resin with the control His-tag. The
> western blot also shows that my protein has His-tag.
>
> Do you have any ideas about my problem? I'm really appreciate all of your
> advices how to solve this. Thank you very much!
>
> My best regards,
> TriNgo
> Sungkyunkwan University
>