Purifying a membrane protein - - the trouble with lipids? (Aug/31/2005 )

Hi,

I am trying to purify a novel membrane protein -

specific activity increases well following an anion exchange column,

I have attempted to concentrate the sample using a vivaspin 10 KDa cutoff filter. As the sample volume decreases a yellow oily layer develops at the bottom of the tube. I assume this to be lipid which has stuck to the colum along with my activity. Has anyone else any experience of purifying membrane proteins this way? Is it possible to get around the co-purification of large amounts of lipid at this first stage?

thanks

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Maybe you will get some idea from the publication:
S. Jindadamrongwech et al. Arch Virol 2004; 149, 915-927

chapter cell membrane preparation

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thanks, that seems a simple protocol that worked well but they make no reference to lipid inteference.

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Hi,
Hope your research is going well. you are lucky to have a membrane protein with a large enought hydrophilic domain that it can interact with your ion exchange column. The lipid that is "co-purifying" with your protein is covering the hyrophobic domains. If you strip the lipid away and don't give it either detergent,lipid, or something else to cover those domains the hydrophobic regions usually cling tightly together and your protein either oligiermerizes or precipitates out. Your protein does not appear to hold the lipid very tightly (unfortunately my current protein has a huge lipid requirement). Hope this gives you another way to look at your protein. You might want to look into the lipoprotein literature to get some ideas also.

Good luck!

mjray

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