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Is AS pp + HIC phisically possible for all proteins?


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#1 paramyosin

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Posted 23 October 2015 - 09:11 AM

Hello all,

As you know, one traditional step after a AS pp is HIC in order to perform a purification removing the high salt concentration at the same time. For this procedure,  you usually add AS to just below the concentration necessary to precipitate your protein and you load the supernatant with your target protein in an HIC colunm....but what happen if your protein is able to maintain itself in the supernatant only at very low AS concentration (for example 20% AS), then, probably this low AS concentration is not high enough to bind your protein to HIC colunm and you can not rise the binding buffer salt concentration because your protein can be precipitated in the colunm.

Then, the this system (AS pp + HIC) is not possible for protein which precipitate at very low AS concentrations, am I right? 

 

Thank you for your coments!!!

 


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#2 mdfenko

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Posted 26 October 2015 - 03:20 AM

if you can precipitate with ammonium sulfate then you can use hic, just run the gradient from a lower concentration.

 

you can also use a different salt (eg sodium chloride) if you want to start with higher salt concentrations.

 

you can download a handbook on hic from ge (and other useful handbooks) at this webpage:

 

http://www.gelifesci...port/handbooks/


Edited by mdfenko, 26 October 2015 - 03:22 AM.

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#3 labtastic

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Posted 26 October 2015 - 11:27 AM

Though ammonium sulfate is best for salting proteins out, you often need something a little more mild for HIC.

 

I ran into this issue at one point and instead used ammonium chloride to bind the protein to a phenyl sepharose column. 

 

Use the hofmeister series to decide which salt to use. You can vary the hydrophobicity of the column material as well (Octyl, phenyl, butyl and methyl) to get things to stick tighter or weaker as needed.

 

Beware though sometimes lowering salt isn't enough to pop your protein off an HIC column...using something hydrophobic may be needed (methanol, ethanol, ethylene glycol, glycerol). As a rule of thumb, HIC can be fantastic for separating things but use it only if other options have been exhausted.






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