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His-tag proteins not eluted from Ni-Beads


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#1 kfj

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Posted 14 November 2010 - 11:58 AM

Hello,

I would be happy if anyone could give me any suggestions....I am trying to purify His-tag proteins (I will use the proteins for making antibodies). I get a quite large amount of protein overexprressed in E-coli, but a majority of the proteins seem to be stuck on the Ni-beads (I use Ni-Agarose from Qiagen) and it is hard to elute the proteins. 0.5M Imidazole does not work very well. 8M Urea buffer elutes a little bit but the yield is not enough for antibody production and I still see a lot of proteins attached to the beads. I am thinking of using 6M guanidin-HCl, but are there any other better ways to elute these "sticky" proteins?? Again the purpose is to make antibody so it is OK to use denaturing methods (I would still try to reduce denaturing agents if possible. The company for antibody production told me that they have successfully made anitbody with antigens dissolved in 5M Urea).
Thank you very much for your help!

kfj

#2 sixerguy98

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Posted 18 November 2010 - 02:03 PM

Are you actually able to get your protein (under native conditions) to stick to the beds or are you only getting it to work under denaturing conditions? If you are under native conditions, how much beads are you using compared to the volume of lysate that you are adding and how long do you bind your lysate to the beads? Do you do this is the cold room or at room temperature? I am asking because I am having a difficult time getting my lysate to actually bind to me beads. What lysis buffer are you using?

I will think about your problem and get back to you..but I have had better results with urea than guanidine solution when it comes to amount eluted from nickel beads.

#3 James W

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Posted 22 November 2010 - 03:06 PM

Just wondering, how do you know you are getting good expression to begin with?

Have you had success using the same method with similar proteins?

Have you tried Promega's MagneHis particles? Might not be much difference but it's worked pretty well for me.

You might also want to try HQ tagged recombinant proteins, I believe these are less sticky than His-tagged proteins.

Other than that, try upping your Imidazole to .75M and vortexing your beads in the elution buffer more vigorously.




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