I am trying to purify a protein..First I run Ni affinity column and the protein was eluted with KPB pH7.9, imidazole 500mM and glycerol ..then I diluted with No Salt buffer containg 50mM TrisHCL pH8 and glycerol 10% After that I run ion exchange chomartography using 2 buffers Buffer A: 50mM Tris HCl pH8, 20mM NaCl , DDT Buffer B: 50mM Tris HCl pH8, 1000mM NaCl, DDT but there is no peak coming. Is there any solution for that problem ?
Dear Sherif,
After running your protein through the Ni affinity column, did you check your eluates for protein bands in the expected position for your protein, and in increased quantity, using a PAGE gel - I would routinely check all 'run-through', wash and eluted fractions, in case my protein(s) of interest failed to concentrate in the expected eluated fraction(s). It was my misfortune, a few years ago, to have a protein 'lose' its Histidine tag during the protein expression step, so that it could not be selectively bound to a nickel column.
If this was your problem, then you will need to use other methods to purify your protein. Please check whether your protein is appearing in your un-bound fractions, and respond, as I might be able to suggest the method that worked for me. If not, I will take a closer look at your choice of binding and elution solutions.
Dear Sir. Concerning your issue about the purification of protien. I think the following below links may help you in your analysis:
https://brf.anu.edu.au/files/protein_purification_handbook_0.pdf
https://worldwide.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/protein-purification-and-analysis/
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/ge-strategies-for-protein-purification.pdf
http://cdn.intechopen.com/pdfs/28765/InTech-Protein_purification.pdf
Thanks
Dear Zaoe Gaffan
I already did the SDS PAGE and my protein of interest appears in the eluted fraction and expression is good.
Dear Sherif,
I am glad to know that your His-tag binding and elution is working. Isam supplied a very comprehensive selection of links, which I am sure will provide solutions to your protein purification problem(s). Although there is quite a lot of material to read through, it is quite simply and clearly presented. Good luck with your purification, I hope that you make rapid progress.
Dear Sherif,
if you look at Isam Elgailani's earlier answer, you will see that he has included 4 links, which I have browsed: they all look very helpful, and the Promega one looks particularly clear and concise.
One thing that seemed odd to me, is that you dilute your Ni-column eluate with 'No-salt buffer', whereas I usually dialyse my eluate overnight, in a [50mM] Tris Buffer, pH8, containing 100mM KCl, EDTA and DTT, to 'exchange' buffers before the next step, in which I cleave the His-tag off, using TEV-protease, performed the next day, then another dialysis step, to remove the DTT , prior to Ni-column Histidine tag removal, as I did not want these in my purified protein, adding more DTT before my first Gel-filtration (Size-Exclusion) column separation.
Ion-exchange column chromatography was my last purification method, before protein concentration, checking fractions for my protein band at each step. One thing to note is that DTT oxidises very quickly - at room temperature it is only effective for about one week so, to protect your protein, it needs to be added to your Ni-column eluates immediately after collection, and to your ion-exchange column buffer(s) just before filtration and use.
I hope this helps you find an answer to your 'disappearing protein' problem.
Zoë.
i see the links now thank you
thanks for you answer
I am trying different buffer now for Ni affinity column hoping that it works
but I will consider your notes
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