Purification of scretory protein from bacteria - (Feb/02/2010 )

I am new in protein work. I am planning to obtain scretory proteins from bacteria and run 2-D gel electrophoresis. How the scretory proteins be purified? and is the range of pH4-7 good enough for doing the IEF of the secretory protein?

kfszelok on Feb 2 2010, 10:24 PM said:

Do you mean you want to purify secreted proteins from the culture supernatant, or you want to purify cytoplasmic or membrane proteins involved in secretion?

If secreted proteins, grow bacteria in culture conditions that will induce secretion, pellet the cells, then filter the supernatant through a 0.22 um filter to remove any leftover cells. Acetone or TCA precipitate the filtrate, and resuspend in sample buffer. pH range will depend on your bacterium. I would start out with a 3-10 IPG strip to see where most of your proteins lie, then adjust the range as needed for better resolution.

fishdoc on Feb 3 2010, 10:02 PM said:

Yes! I am targeting secreted protein! Thanks! It is really useful!
BTW, what is the use of acetone/TCA precipitate the filtrate?

THANKS!