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his-tag prufication lysis problem:(


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#1 mbsea

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Posted 31 July 2012 - 03:15 AM

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.

#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.
talent does what it can
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.
talent does what it can
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.




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