I am working on macromolecular crystallography. Now I am doing purification of my protein. I tried with lysis buffer which includes tris, pmsf, beta-ME, nacl and kcl. I didn't get a proper result. Can anybody suggest something about the lysis buffer preparation?
Every protein has it's own optimal buffer. Membrane proteins also typically require one detergent or a lipid or a combination of several lipids detergents.
Just try many buffers. Nobody can tell you a priori which condition will work out for your protein. Some people try literally thousands of conditions to stabilize a membrane protein. You can minimize your efforts by statistical experimental design.
thanks to all. hope i will purify my protein with your valuable suggestions.
Thanks
Hi Subasri,
there are different methods to extract the protein from the membrane. Some researchers solubilize the proteins directly from the crude membrane after cell lysis. They mix the detergent directly with the supernatant of the low speed centrifugation. In this case you usually need more detergent and your preparation is less "pure". Most researchers collect the membranes (cell lysis, low speed centrifugation, high speed centrifugation) and solubilize the membrane proteins from there with detergents (try non-ionic ones first, like DDM, DM with inner membrane proteins). A good strategy is to fuse your membrane protein to GFP, to enable high-throuput techniques for solubilization studies (screening of detergents). There are freely available books on
Google Books, e.g.: Production of Membrane Proteins: Strategies for Expression and Isolation. If you want to crystallize the protein afterwards, avoid phosphate buffers, as they often produce salt crystals with conditions containing divalent cations. HEPES and Tris are a better choice. As Shruti already wrote, you have to use the detergents above the CMC. For solubilization we use 1% of detergents, like DDM, DM and 2% for beta-OG. For all the buffers after solubilization we use, 3-5 times the CMC. The CMC of detergents changes with salt concentration. For DDM, for instance, the CMC drops with inscreasing NaCl-concentration. A good source for information on detergents is the website of Affimetrix: http://www.affymetrix.com. We sometimes use 5-10% of glycerol in buffers, to increase stability of the proteins after solubilization. Good luck,
Magdalena
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