questions on cycloheximide pulse chase assay - (Sep/03/2011 )

hi, I want to ask several questions regarding cycloheximide pulse chase assay.

procedure:

I want to see if my target protein A extends the half-life of another protein B, so I transfected 293FT cells with B alone (group 1) or A+B (group 2). 48h post transfection, I added cycloheximide at final conc. of 100 ug/ml and lysed cells at 0h, 12h, 24h, 48h with same volume of RIPA buffer. At last, I loaded same volume of lysate on the gel and did western blot.

result:

1, expression level of B in group 2 is much higher than 1.
2, as shown in the figure, ratio of 48h/0h expression of B: group1: 40%; group 2: over 100%
3, B expression in group 1 was decreasing with time, but in group 2 it was even increasing at some time points.

my questions:

1, can I get the conclusion that A extended half-life of B?
2, B expression is severely enhanced in group 2, does it mean A stablized B or A increased the mRNA amount of B?
3, do I need to check the mRNA expression for this experiment?
4, why in group 2, protein level was increasing? very weird.

last question seems to be a very basic one but I got confused here. I want to know if the concentration of protein or DNA/RNA change when they are degraded, partially or completely.
In this experiment, I didnt do protein assay I just loaded same volume of lysate. I used a-tubulin as the loading control and the data was normalized to a-tubulin. Is it correct doing this?

many thanks.

First off, you shouldn't see an increase in protein levels after addition of cycloheximide. Did you confirm that your cycloheximide is working correctly by looking at the level of a protein that you know degrades in a relatively short time frame?

Normalisation is tricky when using inhibitors of protein synthesis - how do you know that your reference protein is not changing? You will probably be better off using total protein concentration.

48 hours, or even 24 hours is a very long half life for any protein, the average half life is less than 12 hours (IIRC).

Cycloheximide does not alter the RNA expression, just the protein synthesis step.

bob1 on Sun Sep 4 00:36:07 2011 said:

thank you very much.

I know the protein level shouldnt be increasing but I just dont know why. I repeated this experiment several times and it always happened. I know cycloheximide doesnt cause changes of mRNA copies but since the expression level of starting point is so different that I suspect mRNA level of B might have been enhanced by A and that is why I want to do a RTPCR to check this.

Actually a-tubulin expression also decreased after cycloheximide treatment, but very slowly in 48h time frame. I used it to normalize the result of group 1 and 2 just to show that the stabilization is specific to B and doesnt affect a-tubulin. In fact the result with or without normalization has nearly no difference.

Regarding the half-life of protein, I also thought it was short before and tried 0, 6, 12, 18h time course but it didnt work. Then I made it longer like this and my labmate ever tried 1d, 2d, 3d, I think that is really dependent on what kind of protein you are dealing with. A new NATURE paper just reported that the half-lives of proteins range from 0 to 1000 h and the median is 46 h, while the median of mRNA is 9h. you can check it if you are interested. "Global quantification of mammalian gene expression control".

Thank you very much.

You could try a RNA synthesis inhibitor instead of a translational inhibitor.

I'm not sure how effective chx is at stopping all of the protein synthesis (though I know it is the common one). You could try a more potent inhibitor such as etoposide.

bob1 on Sun Sep 4 23:58:40 2011 said:

thanks for your reply. I checked mRNA expression after CHX treatment by qPCR. unfortunately I found they are increasing over time. that maybe explained why the protein level increased after CHX treatment but I dont know why mRNA was increasing. Maybe because it is an overexpression experiment and synthesis of mRNA is much faster than its degradation. I set 0h time point at 48h post transfection and at 48h (96h post transfection) mRNA expression was still increasing, is that normal? I think after 72h post transfection degradation should be dominant. maybe that is only the case for protein, not mRNA? anyway it is too complicated and hard to get it all clear.
maybe RNA syhthesis inhibitor is better, do you have any recommendations? thank you very much.

It may well be that the RNA levels are increasing because there is no protein synthesis - hence no RNases around to degrade it. There is also the possibility that some of the RNAs are increasing because there is a cellular response to the cycloheximide, though this shouldn't affect your plasmid based expression levels.

Overexpression from plasmids is quite commonly detected out to over 120 hours in my experience. I don't know about the RNA levels, as I usually just look at the protein.

Actinomycin D is a commonly used transcriptional inhibitor, but I don't know much about it.

thanks. now we turned to check the endogenous level and to see if what we saw in overexpression experiment was just the artifact. I will keep it updated when I get the result, or if I remember:).