Methods to measure a desired protein's half-life? - (Jun/29/2005 )
I would like to know what are the classic or exciting new methods to measure ONE, certain protein's half-life or turnover rate in vivo and in vitro.
I'm picking up some general ideas from reading the literature. Hopefully, there might be an expert out there to offer their advice. I would GREATLY appreciate the confirmation of my various ideas!
Thanks very much for reading,
non life science grad student
*homer voice* Half life of a protein aaah. *end homer*
In vitro is pretty easy. Label your cells for say 1hr with 35S in your media.. ooops don't forget to pretreat cells for about 2hrs with serine/methionine free media.
Your labelling should be in several 100 mm dishes.
Okay, as soon as you add media to all of them.. harvest one plate of cells.. that is time 0.
Harvest cells in 30' intervals until all your plates are harvested.
Sonicate cells..spin to collect PNS an run a lowery.
Add primary antibody to Xug of PNS.. rock overnight 4C. Then add Protein A/G to the mix for 1hr room temp. Spin and collect beads.. Wash beads several times, spinning and decanting each time.
Boil beads in Lammli buffer with reducing agent. Run on gel. Dry gel and expose to film at -80C for X number of days (generally 1-7).
Do densitomitry on the film. Your specific band should decrease in intensity over time allowing you to determine half-life.
*disclaimer*
I've had 2 beers and am remembering this protocol off the top of my head. This is much harder to do than to type.
Okay, I can't fiind the edit button to my previous post.. but there is a big error in it.. I realized it this morning while taking a shower.
Add the media containing the 35S Methionine to your cells for an hour, this is called the pulse.
After 1 hr add fresh media without the 35S to all your plates and immediatly harvest one plate this is time 0. Then you collect the other plates at predetermined time points... 15 min, 30 min, 1hr etc. This is called the chase.
The rest of the previous post is okay.
This would be called a pulse chase experiment.
Add the media containing the 35S Methionine to your cells for an hour, this is called the pulse.
After 1 hr add fresh media without the 35S to all your plates and immediatly harvest one plate this is time 0. Then you collect the other plates at predetermined time points... 15 min, 30 min, 1hr etc. This is called the chase.
The rest of the previous post is okay.
This would be called a pulse chase experiment.
thanks very much for your insight!