His-trap HP column? - (Aug/25/2006 )

Hi everyone,

I am purifying my 6xHis-tagged protein (42kD) by His-trap HP column (5ml) from Amersham, it looks like the column does not work well, I can get a peak, but it contains multiple bands (more than 10) after I ran the gel. I increased concentrtaion of Imidazole from 20 to 35mM but still have multiple bands, 35mM of Imidazole is the highest concentration I can achieve as there is no peak after I increased it to 40-45mM. And I tried different induce condition: different IPTG concentration, different temprature (26-37C), but it never improve.

Is there anyone who has experiences with His-trap HP column? Does Ni-NTA column from Qiagen work better? Or I should change other kind of column?

Thanks for looking and any kind comments.

Hi Rabbit!

Where is your His-Tag?

When it is N-terminal multiple smaller bands might be not completely translated protein-fragments of your protein (bacterial translation differs from eukaryotic, so there are often incomplete translation products).
When you use a C-terminal tag these bands will disappear because the fragments won't get a His-tag.

If you have bands with bigger size it can be that your protein is forming multimers. Maybe you can see them disappear using denaturating conditions (e.g. urea).

Greetings,
Chakchel

Hi Chakchel,

Thanks for your kind reply..

Yes, my his-tag is N-terminal..but obviously there are a lot of non-specific binding as I have a lot of bands (more than 10), some of them can not be explained by truncated protein or mltimers..

Do you think C-terminal his-tag is better? I am thinking of re-cloning my protein..

Thanks a lot..

Hi rabbit!

Well, it depends on your protein... when you have a C-terminal sequence that for example is important for the localization of your protein there could be bad influence of the his-tag.

But at least you will get of bands caused by incomplete translation...

Do you have antibodies against your protein (N-terminal recognition would be good in this case) - not only the his-tag?
Then you maybe can check which bands are incomplete protein or multimers, too...

Good luck after all!

Chakchel

Hi Chakchel,

Yes, my important domain is at C-terminal, so I choose N-terminal from the very beginning.

I only have an anti-histidine antobody! 3 of the bands are histidine positive, so obviously those histine-negative bands should be non-specific binding -- but HisTrap can not get rid of them.

I checked with Amersham, they said HiTrap Chelating is better than HiTrap, do you have experiences with HiTrap Chelating? Anyway I will try it...

Thanks and have a nice weekend.

Hi rabbit!

Sorry, I don't have experience with HiTrap Chelating.

Have a nice weekend, too! And of course good luck and success with this method! Let me know how it worked!

Chakchel

Hi rabbit,
I tried both qiagen and amersham with no success, but dont let discourage you usint thos systems.
I tryed to purify from mammalian cells under different conditions but apparently my protein was too big.
I changed to another system as in both cases I got a lot of unspeciic binding and my protein being eluted with the flowthrough.
sorry if this was not of much help