His tag protein purification problem - (Jun/12/2004 )
I'm doing with an His-tagged protein. Everything's ok until purification step: my protein is contaiminated with other proteins after elution. I'm using Ni-NTA agarose resin of Qiagen. They said we could use b-ME upto 20mM (I'm wordering what buffer we should add it to); or increase salt/detergent or add etOH/glycerol to wash buffer. Has anyone tried this? How about the concentration range of these components you think i should try?
Increase the NaCl in your binding buffer.
Try 500mM even.
I work with a his tagged protein (~43KDa). The protein has absolutely no solubility problem after overexpression from a pET15b in pLys S cells. The protein does not bind to Ni-NTA. It does not bind to anyother column also. But upon denaturation by 8M urea it binds to a certain extent in Ni-NTA. Almost always the protein goes in flowthrough in any column you put. I tried Ni-NTA, CM-Sepharose, DEAE with various pH ranges (8, 8.8, and pH 6.4 for CM Sepharose as well since the proteins theoretical pI is 10.02). I confirmed it by doing a wester blot with anti-his anti bodies in all cases. Can amy body help??
I am new in the bioforum and I am facing problems in expression of a his-tag membrane protein. I transform the plasmid (pET28) with my gene in BL21 but it didn't work then I tried the mutants C41 and C43 and I got lots of colonies then I start expression but nothing can be seen on SDS-PAGE or even western. Do you have any suggestions?
Thank you and I will appreciate your reply
Qiagen also supplies TALON beads (cobalt in place of nickel )which also bind His tagged proteins.
You can try them. Also sometimes equilibrating the beads in buffer before addind to lysate also helps.
I have very less experience with His tagged protein purification. After purifying my protein (30kDa) using Ni2+ resin I still see other proteins in the eluted fraction. One of my colegue demands that by re-purifying those fractions in the Ni2+ resin once again under exactly the same conditions he can clean it up . Is that possible?
Thanks for your suggestions,
rechromatography on the previously used column under same conditions does help sometime (not always). the binding and elution profile of a protein preparation depends not only on the nature of the protein but also on the relative abundance of other proteins. When the protein concentration is too high in the first run, the elution profile is not good. in the second run, the number of other proeins are less and the elution profile is imporoved.
there has been instances in our department where people have used octyl sepahrose chromatography succesviley with improved resolution on the rechromatogram. one protein was purified by ion-exchange before and after a gel filtration step.
sometimes, chagning the sequence of the columns also helps. i used phenyl sepharose as the first step for a certain protein and thereafter a gel filtration; always, there was another band who couldn't be removed by manipulating these chromatographies. I included an ion exchange run ( Q-sepharose) before phenyl and got good purification.
National Chemical Laboraotry,
Hi, I am trying to isolate some proteins with His-tag using a Nickel column. However, I do not want even small quantities of imidazole in my final preparation. One way to do this is ofcourse dialyzing away the imidazole, but since I want to make all of the imidazole go away, this may take too long.
Does anyone know of a way to strip a Nickel column with something other than imidazole. So, for instance, can I bind the His-tagged protein to the column and strip it using Nickel or Zinc or other metal salts? Is there a protocol for this? What are the potential problems/limitations with this approach?
Thanks in advance for any pointers!
I have to purify a His-tagged recombinant protein using an Atherosclerotic plaques's supernatant: that is,I will add the Tagged protein to a supernatant in DMEM, expecting that my protein will bind to a ligand of interest, then, I have to mix this supernatant with the Ni-NTA beads and do the purification, but my question is:
1) Which should be the equilibration buffer for the beads?? (should I made it also in DEMEN, adding NaCl and Imidazole??)
2) And the wash, and elution buffers, shoud they be made in DEMEN??
Or do you hopefully have any other better idea??
Many thanks in advance
i guess here might be the right place for my question
my protein is fusion with his tag at C-term. But after dna sequencing i found only 5 his-tag there, it is supposed to be 6 there. cause there are 6 his-tag in the vector, and the sequence back and forth in the vector is correct. i wonder if it is possible that it is a mistake, but i have 3 same vectors,2 of which have 5 histag and only one has 6 histag. and my protein sequence in the one with 6 histag is wrong, there is a gap in it. So now i wonder if 5 histag is ok for ni-column purification? if it is ok i will just use the one with correct protein sequence and 5 histags. Thanks in advance.
Hi, I am new to the forum, right now working on proetin purification and interaction with DNA. I am facing the same problem in purifing my his -tagged protein (42KD including the tag). I try to do native purification using native condtion protocol from sambrook. Most of the protein dosent seem to bind to the Ni-NAT, as I lose most of it in the differnt wash steps. Also the final elution is also not very clean as I get other bands on the gel as well. I have used LIC vector from Novagen to express the protein, could it be because of the vector or any othe reason. I do my purification at room temperature.
Please do reply, as I have been struggling with this problem for very long now and my supervisior has run out of ideas too.