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after gelfiltration need to concentrate peptide mix

peptide

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4 replies to this topic

#1 johnuknow

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Posted 02 March 2016 - 07:04 AM

Im wondering after producing a concentrated peptide mix, what would the best of concentrating it be?  Specifically if i wanted to get rid of other non protein stuff like nucleotides that are also hanging around.

Any suggestions.

John



#2 mdfenko

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Posted 03 March 2016 - 06:16 AM

i'm thinking that ammonium sulfate precipitation and resuspension may be what you want, if you have sufficient volume.


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#3 labtastic

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Posted 03 March 2016 - 06:44 AM

Do you have a peptide (like 10-50 amino acids?) or a protein (>100 amino acids?) that needs to stay folded?

 

In general though, the best method for concentrating proteins is ultrafiltration with something like an amicon concentrator. However, this method is not useful for separating out smaller from bigger molecules. 

 

If you want to separate out nucleotides from protein, anion exchange (or cation exchange if you protein is positively charged) is most effective. Or binding your protein to an affinity column (e.g. nickel column if your protein is his-tagged) and washing with high salt concentrations (e.g. 2M NaCl).



#4 johnuknow

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Posted 04 March 2016 - 04:11 AM

Thanks guys.  No, its a tryptic digest of a mix of proteins.  I put them through a gel filtration column so i have dilute fractions.

SHL



#5 mdfenko

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Posted 04 March 2016 - 05:16 AM

if you're not looking for biological activity then you can precipitate with tca, although it may not precipitate very small peptides.

 

why do you think you may have nucleic acids present after gel filtration?

 

if not for your assumption that you have nucleic acids present, i would have also recommended ultrafiltration to concentrate your peptides (you can get down to 500 Da exclusion limit of globular proteins and peptides).


talent does what it can
genius does what it must
i used to do what i got paid to do






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