I have tried sonication, but sonicator is not working now, so what reagent should I use to take out the integral protein. I have tried freeze thawing by using liquid nitrogen and 42 C temperature, but for integral protein, I guess freeze thaw won't work correctly.
Digest the cells with lysozyme for 5 minutes (as if you were making miniprep DNA), and then extract the integral membrane protein with a detergent buffer (i.e. 0.5% Triton X-100).
Thankyou Sir. I will try this for the extraction of integral membrane proteins.
Sonication is not a good approach in our experience. But as your membrane protein is likely to be located in the inner membrane of BL21, then you will nevertheless need to break the cells in some way. Instead of sonicating therefore, you can use either a cell disrupter (for large scale) which uses explosive decompression to produce predominantly inside out membrane vesicles and fragments. If no access to cell disrupter, then digest with lysozyme first in an osmotically favourable buffer containing sucrose and then burst the cells with addition of water. There is a procedure for this called water lysis. After lysis, use detergent containing buffer to extract your protein from the membrane (but take care with choice of detergent as some can inactivate the activity of your protein)
Yes, Mary is right, the water lysis prior to detergent extraction gets rid of a lot of proteins (water soluble proteins) and provides a good enrichment in the membrane proteins (extraction of the pellet in smaller volumes without increased viscosity).
Hi Priyanka, just to add to the great comments above, if you generate E coli spheroplasts by using the techniques mentioned by Mary and Jurgen, you can wash them to get rid of non-specific proteins and then solubilize them with TX-100.
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