I am trying to express full-length tau with high yield and purity. I only used SEC to purify my protein since it is not his-tagged. When I did the UV-Vis to check the concentration of the tau protein. There was a high peak at 260 nm, which is around twice than 280nm. I am wondering how to remove nucleic acids and get pure tau protein.
Dear Cheng
i see two different possible approaches:
1) you can digest the DNA with dnase and or benzoase and remove the fragments by sample dilution/concentration with utrafiltration.
If you are worried about the DNAse bpresence, you can buy a his tagged Dnase ( https://www.sinobiological.com/DNASE1-Deoxyribonuclease-I-DNL1-Protein-g-12324.html or https://us.vwr.com/store/product/24712999/dnase-i-protein-human-recombinant-his-tag ) and remove it by IMAC where your protein will be collected in the flow trhrough.
2)If i'm not wrong, Is it the isolettric point of your tau close to 9?
Because in this case you can try to perform an anionic exchange (working for example at ph=7 or 8) https://www.bio-rad.com/webroot/web/pdf/psd/literature/Bulletin_6881.pdf
where theoretically DNA will bound to the coloumn but not your protein.
good luck
Manuele
Hi there,
Ion Exchange Chromatography and/or Heparin affinity chromatography (Heparin HiTrap) should be helpful. Treatment with PEI (polyethylenimine) is also to be thought of.
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/removal_dna/
https://www.researchgate.net/post/How_does_the_heparin_column_work_for_DNA_bound_proteins
I recommend to you using Dnase to digest the DNA,it is much simpler &time saving. Good luck.
I could try DNAse. The question is that I have to remove DNAse again through SEC coz this is also a protein. Yes. The isoelectric point is 8.25, which is positive protein. I have read through the addgene 16316. They said I could try to use acid to purify tau protein. Have anyone used the protocol from Addgene. Could I replace FPLC with SEC?
https://www.addgene.org/16316/notes/#note-425
Also, I think my protein is DNA bound coz SEC can remove DNA according to the molecular weight, right? How to deal with this scenario? I do not think DNAse or other methods could work if my protein is DNA bound. I think I have to start over from the lysate and use DNAse or other reagents, right? Is there a method for samples after SEC?
Hi again,
If DNA /protein complex is purified by SEC it means affinity between the 2 is sufficient to sustain this purification step. Therefore if you want to get rid of the bound DNA you have to propose competitor for the DNA binding site (Heparin) and/or play with the charge beared by the 2 partners (DNA being negatively charged and your protein being positively charged)
Hi Cheng,
Have you tried the ion exchange? However, as you mentioned, the SEC is usually enough to separate proteins and DNA, so you should pay attention to DNA contamination during purification.
Good luck.
I do not have ion exchange column. I just want to use what we have to deal with DNA contamination. How about acid treatment mentioned in the expression protocol? Have anyone used that before? I could purchase a Heparin column if necessary.
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