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Removal of cleaved peptide after Protease cleavage


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#1 marzieh

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Posted 22 June 2015 - 12:08 AM

hello

can i separate my 5.5 kDa peptide from 17kDa cleaved tag using centricon?



#2 mdfenko

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Posted 22 June 2015 - 05:42 AM

maybe with a 10 kDa cutoff if the 17 kDa is globular.

 

but you'll probably have more success with a chromatographic method (affinity chromatography, ion exchange, reverse phase, hydrophobic interaction, maybe by gel filtration with superdex peptide).


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#3 labtastic

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Posted 22 June 2015 - 06:18 AM

Centricons are not ideal for separating proteins based on size. I've had 10kDa proteins not flow thru a 50 kDa filter. And a 28 kDa protein not go thru a 100 kDa filter.

 

Your best bet is probably ion exchange. 

 

Many times we cleave tags off proteins and remove these tags along with the protease by affinity chromatography. For example, if you express your protein with a cleavagable His6 affinity tag....then cleave your affinity tag using the desired protease that also has a His6 tag on it. Then run the reaction mixture over a nickle column. His6 tagged peptide and the protease will stick to the resin, your protein of interest will flow thru. Maybe you could apply this concept to your system.


Edited by labtastic, 22 June 2015 - 07:57 AM.


#4 marzieh

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Posted 22 June 2015 - 07:53 AM

thanks

On the assumption that the 5.5kDa peptide passes through 10 kDa filter, is it possible to get denatured or some percentage of peptide be trapped in the filter?



#5 mdfenko

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Posted 23 June 2015 - 03:46 AM

thanks

On the assumption that the 5.5kDa peptide passes through 10 kDa filter, is it possible to get denatured or some percentage of peptide be trapped in the filter?

almost certainly


talent does what it can
genius does what it must
i do what i get paid to do




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