Has anybody used arginine to solubilize a protein? I am working with some insoluble proteins and I've discarded the use of urea and other chaotropic chemicals to solubilize them. Arginine is working well, but problems come when trying to bind proteins to: Ni-columns (no bind or very low bind) or ionic-exchange columns (it looks like there is unspecific binding and elution, because no resolution of proteins is observed). I realized the NaCl added to the sample improves the binding to Ni-columns, but it is not enough. Some ideas?
Indeed higher NaCl concentrations are useful for the initial purification steps as they reduce non-specific binding of proteins to the affinity matrix and also to each other. However, 1M NaCl seems excessive and will probably reduce binding of your His-tags to the nickel beads/column.
I think your main problem might be your buffer. Your buffer should have a minimum molarity of 50 mM and ideally of 100 mM. 20 mM simply isn't strong enough to buffer the cell lysate. If you decide to use 20 mM again, use a pH meter or a pH strip to test the pH of your cell lysate and you will certainly see that it will be different from your buffer's pH. Also, Ni-NTA works best at pH >7.6 (pH 8 to 9 will give good binding) as the histidines in the His-tags become protonated at pH
What concentration of Arg are you using? Have you tried reducing it to find a happen medium? This reference may be useful:
http://www.ncbi.nlm.nih.gov/pubmed/19167355
Have you tried detergents like CHAPS or lipisomal reconstitution? If possible, you could create a new construct that uses another affinity method such as GST, strep, FLAG, etc.
Thanks for your answer Jason.
At this moment we are working with an arginine concentration of 400 mM, as we have observed a better solubilization of proteins instead of 200 mM as recomended in the article for binding to Ni-NTA. After solubilization, a dialysis is enougth to reduce concentration of arginine to allow the Ni-NTA binding. However, preliminary assays showed that the binding is decreased (comparing with other soluble proteins that didn´t need arginine solublization), and maybe we are overlooking some additive in the binding buffer (actually we are using 20 mM phosphate buffer pH 7 with 1 M NaCl) that could improve binding to Ni-NTA.
Regarding detergents or liposomal reconstitution is a good option, but new contructs are not a good idea (at this moment), as we have all the working flow based on Ni-NTA :)
Nowadays, we are trying to reduce the arginine concentration to solve this problem other ways than dialysis, as arginine is very "sticky" to proteins.
Indeed higher NaCl concentrations are useful for the initial purification steps as they reduce non-specific binding of proteins to the affinity matrix and also to each other. However, 1M NaCl seems excessive and will probably reduce binding of your His-tags to the nickel beads/column.
I think your main problem might be your buffer. Your buffer should have a minimum molarity of 50 mM and ideally of 100 mM. 20 mM simply isn't strong enough to buffer the cell lysate. If you decide to use 20 mM again, use a pH meter or a pH strip to test the pH of your cell lysate and you will certainly see that it will be different from your buffer's pH. Also, Ni-NTA works best at pH >7.6 (pH 8 to 9 will give good binding) as the histidines in the His-tags become protonated at pH
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