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Massive contaminant with TCA/acetone precipitation

TCA precipitation

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#1 zakarino

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Posted 15 February 2012 - 03:12 AM

Dear all,

I have tried to precipitate proteins from plant extracts using TCA/acetone precipitation (10%TCA final concentration). When I resuspend my pellet in PBS and load on a SDS-PAGE after pH adjustment, I can see plenty of discreet bands on the gel with coomassie blue. However, I have a massive unstained band around above 100kDa up to the stacking gel. I cannot remove the proteins from this putative contaminant using ultrafiltration or filtration as it appears proteins are bound to this and are concentrated or removed (filtration). Also, the solution is unbelievable viscous (like tween or glycerol) but this is not due to protein as fairly low concentrated.

Do you know if TCA/acetone precipitation can precipitate polysaccharides or residues of cell wall from plant which could increase viscosity and possible ways of removing them? Would you think high salt content would create such a massive potato?


Thank you very much for your help

Zakarino

#2 bob1

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Posted 15 February 2012 - 02:26 PM

The usual methods of extracting DNA and other polysaccharides all involve salt and a solvent. I am pretty sure that you have precipitated either cell wall components or the DNA out of the cells and this is acting as some sort of molecular sieve, and retaining your protein(s) of interest. There are a number of things you could try to get rid of these - ultrasonication or sucking the solution up and down (may need 50+ repeats) through a fine gauge hypodermic needle should shear DNA, or you could try adding a DNAse such as benzonase. If it is polysaccharide, I am not sure what to do other than to try the sonication or try some sort of cellulase (perhaps agarase might work).

#3 zakarino

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Posted 17 February 2012 - 08:00 AM

Hi,

Thank you for your comments, much appreciated!





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