problem in pelleting bacteria - (Dec/03/2008 )
Dear all
I am doing protein expression in BL21. After expression I harvest the bacteria by centrifugation at 4700rpm for 10min - 1h (unfortunately I have no option for higher centrifugation). I have several different constructs - for most of them 10min are sufficient to get the supernatant clear and pellet the bacteria but for one of the constructs I dont even get the supernatant completely clear after 1h of centrifugation. What can be the reason for that ? How could I solve this centrifugation problem ?
Usually I get ~1,4g wet pellet from 700ml culture. Is that a normal yield, what are your experiences ?
How could I solve my centrifugation problem ?
pET
-pET-
QUOTE (pET @ Dec 3 2008, 04:56 AM)
Dear all
I am doing protein expression in BL21. After expression I harvest the bacteria by centrifugation at 4700rpm for 10min - 1h (unfortunately I have no option for higher centrifugation). I have several different constructs - for most of them 10min are sufficient to get the supernatant clear and pellet the bacteria but for one of the constructs I dont even get the supernatant completely clear after 1h of centrifugation. What can be the reason for that ? How could I solve this centrifugation problem ?
Usually I get ~1,4g wet pellet from 700ml culture. Is that a normal yield, what are your experiences ?
How could I solve my centrifugation problem ?
pET
I am doing protein expression in BL21. After expression I harvest the bacteria by centrifugation at 4700rpm for 10min - 1h (unfortunately I have no option for higher centrifugation). I have several different constructs - for most of them 10min are sufficient to get the supernatant clear and pellet the bacteria but for one of the constructs I dont even get the supernatant completely clear after 1h of centrifugation. What can be the reason for that ? How could I solve this centrifugation problem ?
Usually I get ~1,4g wet pellet from 700ml culture. Is that a normal yield, what are your experiences ?
How could I solve my centrifugation problem ?
pET
I would not stress about this step. How much protein do you need? If you are getting enough from the 1g that spins down, then I'd let whatever bacteria remain in the sup go and move on. The yield will vary based on the time of induction (mid log phase vs. late log phase) and the protein being induced. Don't worry about what is "normal" or why all of your bugs aren't spinning down, I'd just concentrate on getting the protein amounts you need. If this means you need to up your culture volume to compensate for the loss of bugs which don't spin down, what's the harm?
-Cheamps-