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Salting out specific protein

salting-out

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#1 Raygoza

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Posted 23 February 2017 - 02:52 PM

How does one find the saturation level of ammonium sulfate for salting out a specific protein of interest? I've goggled this but all I get is how to achieve a specific saturation level, not how to find the saturation level needed.  Thanks


Edited by Raygoza, 23 February 2017 - 02:55 PM.


#2 labtastic

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Posted 23 February 2017 - 03:47 PM

Assuming you've checked through the literature and can't find anyone who has done this for your protein before, you'll just have to do by step-wise titration. It's impossible to predict with any reliability how a protein will behave during ammonium sulfate precipitation.

 

Start with a 30% ammonium sulfate cut. Centrifuge and assay pellet and supernatant for your protein (activity assay, gel, affinity column, etc). Assuming it's in the supernatant, add additional salt until 50% saturation and repeat. Then 70%, then 80%, etc. It's a bit laborious but if you do it correctly then you only have to do it once to find your answer.






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