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How to concentrate a small peptide < 2 kDa?

concentrate peptides low MWCO small peptides

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5 replies to this topic

#1 schaapje_86

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Posted 01 October 2012 - 02:58 AM

I have expressed a small peptide attached to a GFP protein. Now I want to cleave off the GFP protein by making use of the TEV cleavage site in between these two proteins. Since the GFP has a His-tag, I want to get rid of the cleaved sample by trapping the His-tagged GFP protein onto a His-trap column. The small peptide will be washed out with the first flow through. Then I would like to concentrate my peptide. However, since the peptide is only 1.7 kDa I have no idea how to do this? Spin columns don't have a low enough MWCO..

Can somebody help me?

Thanks a lot!

#2 drwho

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Posted 01 October 2012 - 04:11 AM

Hi I have tried this dialysis method with good results:

http://www.piercenet...?fldID=04010160

But it goes down to 2kda so may not work out for you, their may be others manufactures out there who may go smaller.

You could also try freeze drying you protein then re-suspending it any volume you want, there is literature out there which says this can alter your protein and so affect function but I never had this issue.

Hope this helps

#3 mdfenko

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Posted 01 October 2012 - 06:18 AM

spectrum sells dialysis membranes with molecular weight cutoffs as low as 100-500 Da.
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#4 schaapje_86

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Posted 01 October 2012 - 07:19 AM

To mdfenko: how would that work? I would still retain all my proteins in the dialysis bag?

#5 mdfenko

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Posted 02 October 2012 - 05:19 AM

To mdfenko: how would that work? I would still retain all my proteins in the dialysis bag?

yes. globular proteins larger than 500 Da would be retained. i think that non-globular proteins would also be retained by this membrane.

you can dialyze against solid peg 8000 to concentrate the protein in the bag. the peg should not be able to enter the bag in significant quantities.
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i do what i get paid to do

#6 peak99

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Posted 11 October 2012 - 07:35 AM

you really had two questions......clean up and concentrate.



separate with a concentrator, eg <2Kda http://www.biocompar...ors-2-000-MWCO/(they may have lower?).

Your protein will be in the filtrate, while all the 2kDA+ will be in the retentate. To concentrate, mdefenko's plan sounds good.



To mdfenko: how would that work? I would still retain all my proteins in the dialysis bag?

yes. globular proteins larger than 500 Da would be retained. i think that non-globular proteins would also be retained by this membrane.

you can dialyze against solid peg 8000 to concentrate the protein in the bag. the peg should not be able to enter the bag in significant quantities.






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