Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

His-tag protein purification - (Sep/21/2001 )

can someone please help me out. i am trying to purify a 125kD His tagged protien using Ni-NTA matrix. i am facing several problems-

1. a lot of non specific protein binding. tried washing with glycerol(30%), 20mM beta mercaptoethanol, and upto 80mM imidazole.

2. very low elution of the protein. even high concentration (1M) of imidazole does not help. on loading the matrix on SDS-PAGE i see a significant amount of my protein adhereing to the matrix.

3. due to the large size of my protein i see very low expression and this too adds to the problem.tried all the tricks the qiagen manual suggests.running out of ideas.

can any one help.

maitreyi

--

Using EDTA (up to 100 mM) you will be able to strip your protein from Ni-resin. The problem is that you will not be able to reuse the resin what you can normaly do after using imidazole-elution buffer (maybe recharging is still able but it will be quite difficult). Be sure that there is not too much DNA in your sample (treatment with DNase is very useful) because it can interact with purificatin and elution. You can also lower pH to 4.0 if your protein allows you to do this.

Good luck!

--

Your matrix is damaged. The resin non-specifically adsorbs protein when the beads in the Ni-NTA column are degraded. Also, we suggest you include a ammonium sulfate precipitation step in the procedure prior to loading on to the Ni column. If you are unclear on how to do this, feel free to email us.

Good Luck

AjoyMycobactexperts, IncA Division of DSTF-Global, LLChttp://www.dstf.bigstep.comdstf@doctor.com

--

Hi, Maitreyi:I have the same problem with yours. My protein is 60KD, and I use Quigen's Nickle-agarose. I can't elute my protein even with 300mM NaCl and 2M imidazole. And I also use ammonium sulfate precipitation to deduce the proteins I don't want, but still the same. Maybe DNA is one of the problem. Have you solve your problem? Can you help me to solve mine?

Ying Luo
Attached File

--

I am having the same problem...and tried all the tricks also. Have you figured something out?

--

:lol: Can anybody tell me the composition of imidazole-elution buffer ( used to remove bound imidazole from Ni-NTA agarose beads) and also how to use it?
Thanks

-mdsr-