Although two ion exchange chromatography ( CEX and AEX) is in place in bind and elute mode ( NaCl conc. < 200 mM in both case for elution) still DNA removal is not sufficient and it is more than 10ng/dose of that product which is not acceptable ?
Is there any specific chromatography , filter membrane or other methods which removes DNA without affecting protein yield and in cost-effective manner ?
If any DNAase / Benzonase is used then how to remove the same after subsequent step ?
Share your views researchers...
Thanks.
There is a very old school method for removing DNA from protein solutions. It is so easy and works so well that I am always surprised that I never see it mentioned in Materials & Methods sections anymore.
The method is PEI (polyethyleneimine) precipitation. In essence, you add PEI to your protein solution to a final concentration of ~0.02%, stir on ice for a half hour while the nucleic acids are precipitated from solution, centrifuge to remove the precipitated material, and you're done! The supernatant can then be used immediately for whatever method of chromatographic purification you prefer to use.
For a sample protocol, simply search Google for "PEI precipitation." I use it to get rid of DNA whenever my purification involves anion exchange chromatography, and it works wonderfully!
IMHO DNase could bind to Blue Gel ( http://www.bio-rad.com/es-do/product/affi-gel-blue-gel )
In general, DNA should precipitate with 20% saturation of ammonium sulphate.
Have you tried hydrophobic chromatography? DNA as it is highly charged should not bind, so if your protein binds, that should work. Or some affinity chromatography.
Size exclusion chromatography should work, but has quite low capacity.
I am totally agreed with Thomas. You can try ammonium sulphate cut to concentrate your protein and removal of DNA from the protein solution as well..or you can give treatment of DNAse during the time of lysis. Add protease inhibitor, RNAse and DNAse all together in your lysis buffer before sonication or whatever method you use for the cell lysis..
Good luck..
There is a very old school method for removing DNA from protein solutions. It is so easy and works so well that I am always surprised that I never see it mentioned in Materials & Methods sections anymore.
The method is PEI (polyethyleneimine) precipitation. In essence, you add PEI to your protein solution to a final concentration of ~0.02%, stir on ice for a half hour while the nucleic acids are precipitated from solution, centrifuge to remove the precipitated material, and you're done! The supernatant can then be used immediately for whatever method of chromatographic purification you prefer to use.
For a sample protocol, simply search Google for "PEI precipitation." I use it to get rid of DNA whenever my purification involves anion exchange chromatography, and it works wonderfully!
longer discussion here:
https://www.researchgate.net/post/How_to_remove_bound_DNA_during_protein_purification
have u tried using a strong anion exchange resin..??
how many washes u tried by increasing and decreasing salt concentrations? like wash wash 1 & wash 2...etc
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