In article <35EAA742.167E at imb-jena.de>, Alan Tunnicliffe <atunni at imb-jena.de> wrote:
:Hi all
::We currently have a protein solution which is 99% (plus) pure,
:however we have discovered protease activity within our sample.
:This protease activity can be prevented by addition of EDTA
:(metallo-protease?),
:and using several protease inhibitors.
::The question I have, is how can I discover if the protein we are working
:on
:has intrinsic protease activity (it is not supposed to) or if the less
:than
:1% impurity is infact a protease ?
::We have tried silver stained gels
:Isoelectric focusing
:SDS gels
:and Native gels to see if we can detect the contaminant.
::Any advice or help would be greatly appreciated
The amount (and pattern) of proteolysis will not change upon further
purifications (using separation techniques different from the ones
already used) if the activity is intrinsic, and will of it is exogenous
protease. Also, if PMSF-like substances are inhibitory, you can use
radioactivly labeled probes to see if they covalently bind to
your protein.
- Dima
