protein half-life assesment - (Oct/29/2013 )

Hello,

I do not work much with proteins and I do not know how the experiment with establishing protein half-life should be done.

I work with HEK293T cells. I want to measure the half-life of my truncated protein which is expressed from plasmid (overexpression). In cells there is also a Wild-Type variant of this protein (slightly longer) with low level of expression.

I did a transfections with wild-type plasmid for overexpression of WT protein and separate transfection with plasmid expressing truncated protein.

After 48h, I added a cycloheximide in conc. 0.1 mg/ml to see the difference in  WT and truncated protein stability.

I made 2 time point, 4h, 8h, because I don't know what is the half-life of my WT protein. Maybe a longer time is needed?

When I did a western blot, i did not see any changes in stability of each proteins, though, I did not have a good control of protein with short half-life.

My main question is, whether it is OK to load the same amount of protein (~30ug) for each time-point, no matter how many cells has survive this experiment, (after 8 h the cells are less viable). Should I somehow normalise the loading amount of protein to the total cell number? How?

What kind of controls should I include: protein with long half-life like: GAPDH?, any others?

and protein with short half-life: like tp53, maybe some others, with half-life less than 4h?

Waiting for reply,

Alchemic

100 ug/ml Chx is a common level to use, but you should titrate it for your system to ensure that it is working - you can do this by looking at the half-life of a known protein.  tp53 is really short - about 30 min, so can be hard to do as you need to do lots of early time points, but is well defined in the literature.

I would normalize to cell number - to do this, lift the cells as you would normally if splitting the cells, collect and spin down.  Resuspend cell pellet in PBS and count the cells.  Then lyse in a defined volume of lysis buffer per cell.  I tend to use 10^4 -10^5 cells/ul for this.  Loading based on protein means that you are saying that all the proteins in the cell are stable, and as such artificially elevating the amount of protein present at later time points where many proteins (if not all) should have been degraded to some extent.

It is also a good idea to do some early time points - I would do 0 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours.  3 time points (assuming you also did a 0 or untreated) is not really enough to get any good idea of how long the half-life is.  You may also want to think about pairing samples - i.e. having the same transfected cells with no Chx at each time point to compare levels of expression.

Thanks for your reply! It was really helpful.

Does this mean that in each time point I should have different volume of lysis buffer? Should I load also different volumes per well, e.g. 5% of total volume, so the amount of protein from the same number of cells?

Does anyone knows other proteins like tp53 that have relatively short half-life?

Yes, you will have a different volume of lysis buffer at each time point, but that isn't important as you want to load a standard amount of cells.  For many proteins 100,000 cells is enough to see the protein easily on a western blot, so if you lyse at 10^4/ul all you need to do is load 10 ul.