293 cells sonication - How do I do it???? (Aug/14/2006 )
I made a transfection of 293 cells with a plasmid and now i need to see the expression of my enzyme... but i dont know how make the sonication...
anyone have an idea?
Do your lab have a sonicator?
If yes, ask someone in the lab to show you. It is a quite dangerous equipment as it may damage your eardrum .
Just extract ur cells in ur lysis buffer. Introduce sonicator into the extract (in a tube) and sonicate briefly for 1-2 seconds. use ear covers.
U could also leave out the sonication part, if ur protien is abundant.
If you use a probe sonicator then heating of the solution may become a problem . I always sonicate my cells at between 8-10 Amplitude Microns on ice for 5 seconds, 2-3 cycles over a 90 second period. Look at your sonicated suspension under the microscope to make sure sonication has been successful. Also in your mix add protease inhibitors to reduce proteolytic attack.