Can I use methanol fixation to fix cells before proteomic analysis? - (Feb/11/2014 )
I am working on a project that requires me to apply a probe to cells to globally modify proteins and install click chemistry handle before pulling them down. This will be followed by protein identification and comparison in protein abundance among samples using MS/MS. However, I found that the probe turns out to be toxic to cells (especially since it is slow and needs to be incubated with cells for a long time). I want to fix cells with cold methanol, but not sure if that will affect downstream analysis (ie. loss of proteins or adduct formation with proteins). I wonder if anyone has any experience or comments about this. Thank you.
This is what I understood from your post (correct me if I am wrong)-
You have an azide or some toxic probe which you want to put in your cells and then by using click chemistry you want to pull out the proteins. Right?
Now my question is, if your probe is toxic, and you will fix the cells with methanol before adding the probe, how will it bind to the proteins?
With methanol, you'll definitely loose proteins. Can't your probe be converted into some other functional group which is not toxic to your cells like biotin?