In a project, I am required to degrade proteins to a size of 10Kd. This would prevent protein from passing through ultrafiltration and being absent in the resultant. I suggested using SDS, however, I have been advised that SDS will not degrade proteins small enough.
In my lab, I was told to use a strong acid to hydrolyse the peptide bonds, but i think this will interfere with plastic polymers that i am trying to study
Any suggestions
SDS will only unfold proteins, not degrade them. To cut up protein into smaller pieces, you might use a mixture of proteases, digestive enzymes that hydrolyze peptide bonds in a (more or less, depending on the specific protease) sequence-dependent manner.
Hi Aidan,
Could you clarify if you need the peptides to be 10kDa or simply smaller than 10kDa? You only need them to be degraded then a commonly used enzyme to degrade protein when purifying nucleic acids is proteinase K. Another option is trypsin. This is commonly used in mass spec protocols.
Dear Adain
ass Annemarie told sds is not able to degrading proteins but just to unfold.
Generally the best way to fragment proteins iis use a proteases as trypsin but of course you will add a protein contaminant with a mw>10 kda to remove the other proteins and this is a partial solution.
to solve this problem you can try to add an his tagged protease..that you can remove afterwards by absorbtion on magnetic beads for imac
orherwise probably you can try chemical digestion
https://www.ncbi.nlm.nih.gov/m/pubmed/18429274/
but i dont know the nature and mw of your polimers and therefore you have yo evaluate which chemical reagent is able to produce fragment of the size that you need and if the polimers is stable in this conditions.
just one question: what about remove proteins by precipitation (ph acidification with ammoniun acetate as happen in plasmid kit, ammonium sulfate, pei) instead ultrafiltration? this conditions will lead also to the precipitation of your polimer? because in this way you can certanilly pre-treat the sample with a protease and precipitate everiting.
good luck
Manuele
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