We are treating cancer cells with inhibitors to see if we can inhibit the Ras-Raf interaction. I can clearly detect the interaction via coIP but I am having trouble seeing inhibition of this interaction. I then read that some people switch their cells from 10% to 2% FBS-containing media before doing these sorts of experiments. Does anyone know the point of this?
Point is to prevent activation of the cascade with proteins from FCS. I used to work on RhoA and MAPK, 24h prior to experiment I would transfer cells in serum free media. I am not sure that this would work good for your experiments, since you are testing inhibition.
Thanks for getting back to me. I had thought it would be that, but people do the same for inhibition experiments as well. Force of habit, I suppose? Oh well, thanks! Much obliged!
I think they do to avoid the activation of RAS/RAF pathway by growth factors present in FBS. You can try 10% FBS, 2%FBS and serum free medium to analyze the effect of inhibitor on RAS/RAF interaction.
I get used to perform starvation for hormone induction. Start
1.washing with PBS
2. İ ncubate for 2 days with 2%charcoal treated FBS
3 change the medium with 0.5 % charcoal treated medium for 1 day and start induction .hormone addition also start at this point.
The switch is to reduce serum contained growth factors (EGF,FGF,HGF etc), that may stimmulate RAF-RAS cascade. To see induction you should starve to get basal levels down, but to see induction I would try without starving to have a high basal level. The problem is the read-out. Different growth hormone pathways and feed-back loops converge on e.g. ERK. If you measure P-ERK as a read-out, your inhibitor may inhibit one branch of the cascade but serum may stimmulate an other one. So you can't see effects. Try to get an read-out as high in the cascade and as specific to your inhibitor target as possible to avoid this.
GOOD LUCK!!
Thanks everybody for the answers! I had already seen a read out in terms of down regulation of phosho-Erk and -Akt and reduction in cell proliferation. It was just the mechanistic Raf-Ras coIP that I was having trouble with. I made a few adjustments to my protocol, switched antibodies and I got it working great now. I think for inhibition having a high basal signal is a good idea, so we didn't do the serum starvation.
Cheers!
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