How to prepare membranes from E.coli? - (Jan/15/2008 )

I need a to prepare membranes from E.coli (BL21) in order to check whether the expressed protein is asosiated with the membrane.

The background of the story is:
I do see a fair large amount of protein in the pellet (insoluble fraction) which either can be in inclusion bodies (most likely) or protein assosiated with membranes.

In order to check this, I want to prepare membranes (outer membranes and organell membarnes). The preparation method should be gentle so that assosiated proteins are not detached from the membane.

Later I would like to check for the localisation of my protein in a) the soluble fraction membrane fraction c) insoluble/pellet fraction.

The protein definetivly has no membrane anchor or transmembrane regions so that it only can be loosey assosiated to the membrane.

So what I need is a protocol to prepare E.coli membranes in a gentle way that this interaction is not distirbed during the preparation process.


And as additional information: of course I try in parallel urea extraction of the pellet. But refolding this protein has turned out not be be straight forward. Therefore I would like to give the above mentioned experiment a try.

THX for your comments :-)

I had to do this myself and it is a PAIN. I had to sonicate my cells then do a multi-gradients ultracentrifugation sucrose seperation. There are some good papers from the 1970s and 80s that go through this. In fact, I think there is one entilted isolation of outer membrane proteins from E. coli.

It may be easier to isolate the inclusion bodies first and rule those out. Do the membranes last!

I need a to prepare membranes from E.coli (BL21) in order to check whether the expressed protein is asosiated with the membrane.

The background of the story is:
I do see a fair large amount of protein in the pellet (insoluble fraction) which either can be in inclusion bodies (most likely) or protein assosiated with membranes.

In order to check this, I want to prepare membranes (outer membranes and organell membarnes). The preparation method should be gentle so that assosiated proteins are not detached from the membane.

Later I would like to check for the localisation of my protein in a) the soluble fraction membrane fraction c) insoluble/pellet fraction.

The protein definetivly has no membrane anchor or transmembrane regions so that it only can be loosey assosiated to the membrane.

So what I need is a protocol to prepare E.coli membranes in a gentle way that this interaction is not distirbed during the preparation process.


And as additional information: of course I try in parallel urea extraction of the pellet. But refolding this protein has turned out not be be straight forward. Therefore I would like to give the above mentioned experiment a try.

THX for your comments :-)