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Protein precipitation with TCA?


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#1 Juliasarmoire

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Posted 15 November 2010 - 12:06 PM

More and more annoying questions ;) I need to get my protein concentration higher, it's currently only 500 ug/ml and it needs to be at least 1 mg/ml. I have used Millipore filter devices before, but somehow my protein didn't like it and I ended up losing pretty much 70% of the protein sample in the process. I'm now trying to use TCA, but keep reading some horror stories about it too... so I was wondering is precipitation the protein with TCA ok? And how much you normally manage to get out by using it (hopefully more than 70%). I'm a bit desperate, like always ;) And what is the best TCA protocol or the one which has at least worked for someone?

I plan to dissolve the protein in the end to 8M urea... so in theory it should hopefully dissolve.

#2 protolder

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Posted 15 November 2010 - 10:35 PM

Hola, I donīt know why you loose high amount of your protein with ultrafiltration, the cutoff of membranes isnīt exact and some proteins of little higher MW cross them so you have to choice a membrane with a cuttoff enough minor than your protein and always analize retentate and filtrate. There are 3KD and 10KD cutoff. TCA isnīt a good method to concentrate your sample because the protein is denatured and difficult to redisolve, it is used to precipitate radiactive proteins or diluted samples to apply in PAGE.OTher possibility is amonium sulphate pptation, or using dialysis tubing. in the first add ammonium sulphate to a 100% saturation (see the tables in google) centrifuge, dissolve the pellet in the minimun volume and dyalize. But you have to notice that the cutoff of normal dialysis tubing is about 15 KD. If you find a compatible dialysis tubing you could pack your protein solution and put the bag on a paper towel or any powder sephadex which will absorb the sweat of the bag untill the volume be the wished. The final option is liofilize and disolve in the minimun volume knowing that the salts of the buffer are concentrated too. Good luck

#3 K.B.

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Posted 16 November 2010 - 10:18 AM

Ammonium sulphate is good if you don't mind salt in the sample - for some application it may not be acceptable. I like acetone precipitation - it's fast, reliable, acetone is easy to remove as it evaporates quickly, and I never had problems with solubility afterwards - but take a note that it may depend on your sample. For me it was also important it removes at least some of the organic-soluble impurities.




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