Yes!

membrane from mammalian cell is very different to Bacteria (Gram positive and negative too), specially structurally.

For each cell depending the localization of the interest protein is necessary a different procedure for solubilization.

The difference comes from the strength of your detergent and the hydrophobicity of the proteins you are looking for. If your using SDS based buffer then the difference is marginal. But a urea/CHAPS buffer for 2-D and your going to see a difference. There are several protocols from manufacturers of lysis beads for bacteria that are considerably more efficient than a mortar and pestle. they also allow you to work with smaller volumes with less loss. Mammalian cells will typically be easier to isolate if from culture rather than tissue as well. We usually use a dounce homogenizer in a hypotonic buffer with protease inhibitors. Once nuclei are removed the membrane fragments are collected and solubilized in a buffer that is compatible with the downstream assay.

The real test is, however, how are you going to detect/measure your protein of interest to determine how successful you isolation was? many (most) membrane proteins have some form of post-translational modification that can interfere with stains or blots. If your target is unknown I would suggest picking out a known protein that is highly conserved and compare by blot and different staining methods until you are satisfied with your isolation.

Cold spring harbor has an excellent resource ISBN-10: 0879697873 | ISBN-13: 978-0879697877

Should be about $100 USD from amazon.

Thank you for your advice. I have not expressed the protein in bacteria and don't know really how it behaves. I am going to express a receptor and purify it with affinity chromatography and after that analyse the purity with western. What are our suggestions based on this? Can I start trying with solubilization buffers used when extracting proteins from procaryotic cells and optimize from there?

I am not going to lyze the lyze the cells with a buffer ( Tris, EDTA, Tritonx-100 and protease inhibitors ) and not mechanically, is that a problem? Should I do it mechanically?

Eukaryotic membrane proteins are usually expressed in a baculovirus system, which has the machinery able to address and deliver membrane proteins. Very often, but not always, expressing membrane proteins in bacteria will lead to inclusion bodies (aggregates of protein). What do you know about your protein? Do you know its sequence? Is it predicted to be glycosylated? If so, you might have a hard time expressing it in bacteria.

>membrane proteins are solubilise in triton X-100, CHAPS, Phenyl thiourea, SDS

>CHAPS or Triton X-100 good for membrane protein solubilization

>the combination of Triton X-100 and Phenylthiourea also good for solubilization

I am not considering expressing the protein i bacteria or in any other cell than HEK293.

Oh, sorry, I obviously read too much into your original question ... We have had success with Triton-X 100 and CHAPS detergents when isolating membrane proteins from HEK 293 cells. It is really protein dependent though to find a detergent suitable to stabilize your particular protein, so we have done this by trial and error in the past to find the right detergent at the appropriate concentration. If there is some function you can monitor, that helps.

Thanks Maybe my question was writen in a bad way. So the solubilization is more protein dependant than cell dependant.

Anette,

You have chosen a very challenging path. Isolating a receptor by it's ligand using affinity chromatography is dependent on two things: the paired affinity and the structural requirement of the receptor to maintain a functional binding site. To make things easy I would test "off the shelf" detergent mixes like RIPA buffer from Santa Cruz (http://www.scbt.com/datasheet-24948-ripa-lysis-buffer.html) and do an IP with beads conjugated to your ligand of interest available from invitrogen (http://products.invitrogen.com/ivgn/product/14311D?ICID=search-product). You could use this system to screen buffers that will solubilize the receptor from the membrane and still retain it binding ability. Remember, however, that your final prep will not be pure receptor protein since the conditions of solubility will not likely dissociate other membrane components from you receptor. I think of it as pulling a weed from the garden. the roots hold a lot of dirt and other plants that come out with it. Oh, and yes a bit of mechanical disruption will definitely help give gentle detergents a chance to interact with membrane proteins. In the end I really like beta-octylglucoside based buffers for IP.