his-tag prufication lysis problem:( - (Jul/31/2012 )

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.

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.

many thanks. but i have no idea how to do it:(

you can remove the chelators by dialysis.

you can sequester them by titrating with nickel.

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.