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protein aggregation problem


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3 replies to this topic

#1 anindya

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Posted 15 July 2009 - 03:30 AM

i add TritonX-100 to make my protein soluble before sonication.upto elution i maintain 2% TritonX in the buffer.But i need to get rid of TritonX 100 during dialysis,i add 2mM beta mercapthoethanol but that doesnt help.when i start removing triton x the proten precipitates out.
any suggestion?

#2 archit

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Posted 14 August 2009 - 11:45 AM

you could try with a higher concentration of b-Mercaptoethanol or with a suitable concentration of DTT.

#3 miBunny

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Posted 14 August 2009 - 06:11 PM

Triton X is not very easy to dialyze out because of its low cmc. I think Pierce offers some kits to remove triton-x.

The problem is that a lot of proteins require the detergent to stay in solution. If you remove the detergent, the protein precipitates out of solution.

Can your downstream application handle a different detergent? You could do a detergent exchange.

#4 DaveD

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Posted 11 February 2010 - 09:39 PM

I agree with the previous poster. So, how about avoiding Triton altogether?
The reason why your protein is aggregating is likely that the buffer is not compatible with your protein (or the other way around :)
So, one way to figure out which buffer works better would be to establish first the buffer that keeps your protein solubilized and then incorporate that buffer into your protocol.
How would go about finding the proper buffer? You could apply a solubility optimization screen kit from Dilyx for that purpose. You'll go through more than 90 different solutions and figure out which ones work and which ones aggregate your protein. If you're lucky they could sign you up as a beta tester and you'd get to test a kit for free.

My 2 ct
DaveD (Dilyx Biotechnologies)




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