Help:Protease/amylase seperation

ferro at msvax.mssm.edu ferro at msvax.mssm.edu
Thu Nov 9 06:49:45 EST 1995


I would start to try a longer incubation time of your prep with
subtilisin. This way the contaminants will be much lower,
and the overlap during Superose 12 will be reduced giving
you a good chance to get pure amylase peaks.
If it doesn't work, you should try a FPLC ion-exchange
column.
hope it helps
emer





In article <47q6mi$k74 at mserv1.dl.ac.uk>, Cormac Shaw <CSHAW at acadamh.ucd.ie> writes:
>Hi there,
>                       I wonder if any of you could help me with a small
>protein seperation problem.
>As part of my study of the domain structure of an amylase, I have 
>been attempting to partially hydrolyse the enzyme and establish the 
>size and biological activity (if any) of the hydrolysis products 
>using subtilisin (Boehinger-Mannheim).
>                       The problem is seperating theprotease from the 
>amylase components after digestion. Gel filtration (on Superose 12) 
>gives me a protein elution profile which just shows a complex mixture 
>of subtilisin components and some other tiny trace peaks which are 
>presumably from the amylase but they're all mixed up together. The 
>literature contains similar seperations but they all to seem to have 
>nice clear enzyme peaks with no sign of the subtilisin at all - 
>what's their secret (could it be that they just have hugh excesses of 
>their enzyme? My levels are unfortunately pretty low).
>        I am considering trying to remove the subtilisin by affinity 
>chromatography (using Bacitracin-Superose 4B) but this looks messy 
>and I'm doubtful if it will remove all the subtilisin components - 
>the protease seems to breakdown itself during incubation. Any 
>suggestions as to simpler / quicker methods would be greatly 
>appreciated.
>
>Thanks for reading,     C.
>________________________________________________________
>Cormac Shaw, BSc. / cshaw at acadamh.ucd.ie / Dept. of
>Industrial Microbiology, University College, Dublin 4,
>Ireland.
>[Insert humourous latin/phrase of your choice here]



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