Hi all; I finally cloned my gene of interest and expressed it. but i could not find an appropriate lysis buffer. the lysisi buffer that qiaexpressionist recomend for native proteins, does not contain tris and edta. Although i added lots of lysosyme, i could not manage to lyse the bacteria. even by using sonicator. But i can lyse my pelelt by using lysis buffer contains edta, egta, tris.Did anyone try lysis buffer for ni nta prufication that contains this ingredients. I know they are not recomended for ni nta system but i could not find any buffer that i can lyse the cells and my protein shows activity. I will appreciate your help.
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#2
mdfenko
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Posted 31 July 2012 - 05:09 AM
the edta and egta will strip the nickel from the ni-nta column.
you will have to remove or sequester them before applying the lysate to the column.
you will have to remove or sequester them before applying the lysate to the column.
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
i do what i get paid to do
#3
mbsea
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Posted 01 August 2012 - 01:53 AM
many thanks. but i have no idea how to do it:(
#4
mdfenko
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Posted 01 August 2012 - 07:26 AM
you can remove the chelators by dialysis.
you can sequester them by titrating with nickel.
you can sequester them by titrating with nickel.
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
i do what i get paid to do
#5
Missle
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Posted 29 August 2012 - 12:13 PM
EDTA would be a problem however I routinely use a Tris based buffer (50mM or 100mM Tris, pH 7.5) and have no problem with subsequent binding to nickel columns.
