I am working on an actin-like protein which is supposed to form filaments. However, I noticed that the filament formation in my case was very poor, whatever the buffer I used (usually, 50mM Tris 7.5, 100mM KCl, 2mM ATP and 4 mM MgCl2). I believe this is mainly caused by the fact that the protein purifies with nucleotides from E.coli. I already have 1mM EDTA in my Gel Filtration buffer but this is not enough to remove the potentially bound nucleotides (ATP/ADP or GTP/GDP). Do you have any ideas? Is denaturation and refolding the best way to remove this tightly bound nucleotides ?
I usually apply this protocol to remove nucleic acids, but this should also work for nucleotides (because most of the binding comes from phosphates) :
If your protein is His-Tagged, just bind your protein to the resin, wash with your favourite buffer without imidazole, wash a second time with the same buffer at high ionic strength (no imidazole and 1 M NaCl) : your nucleotides should elute from the column. Then wash again with the first buffer to lower salt concentration, and then elute with imidazole.
Hope this will help
Don't know if you'd achieve separation, but you could try ion exchange. This works for nuleic acids - not sure about individual nucleotides though.
I usually apply this protocol to remove nucleic acids, but this should also work for nucleotides (because most of the binding comes from phosphates) :
If your protein is His-Tagged, just bind your protein to the resin, wash with your favourite buffer without imidazole, wash a second time with the same buffer at high ionic strength (no imidazole and 1 M NaCl) : your nucleotides should elute from the column. Then wash again with the first buffer to lower salt concentration, and then elute with imidazole.
Hope this will help
The protocol below is your best bet but potentially creates a refolding problem, otherwise a q-column as suggested previously may be an option
Article Rapid and Efficient Purification of RNA-Binding Proteins: Ap...
(1) Precipitation of the protein with ammonium sulfate may help. It may be necessary to repeat the procedure a few times. (2) Depending upon the affinity of the nucleotides, you may be able to remove them by diluting the protein well below the Kd, then reconcentrating by either ultrafiltration or column binding.
Thanks for all your comments. I will try High Salt during purification and in last option the refolding. I know refolding can be difficult but these actin like proteins are usually very well folded.... Renaturation should be ok-ish. I will tell you how it is.
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