I work in plants and I am following a protocol to do proteomics. However, I cannot get enough protein to do any of the downstream steps. Everytime I get to the acetone precipitation, the protein remains in the acetone! It shouldn't be possible and I am starting to go crazy. Any help is appreciated.
We harvest the root tissue and immediately freeze in liq. N, then grind the tissue using a bead beater in the cold room extraction buffer, let sit on ice for 1 hr, and spin down the crude lysate at 18,000xg for 10min at 4C. The supernatant (lysate) from this step is full of protein, while the pellet has very little. This is good, everything going according to plan.
We then transfer the supernatent to a new tube and add 4x volume of -20 chilled acetone. Vortex the samples and let them sit overnight in the freezer. The next day, we centrifuge at 18,000xg for 10 min at 4C again. There is a while smear in the side of the tube that we were (incorrectly) assuming was protein. We discarded the supernatant from the acetone spin and resuspended the pellet only to perform a BCA and find there was no protein present.
We have tried a new protease inhibitor. Made sure to keep everything on ice. Tried fresh reagents, and spun for longer. Nothing is working.
Eventually, we had the idea to dry the acetone supernatent we had been discarding. Low and behold, there was about 60% of our protein.
Has anyone encountered anything like this before? I inherited this protocol from an old grad student who never had any of these issues. Any suggestions?
We are trying to do a special downstream protocol on the proteins, so unfortunately I can't use a kit or other extraction method.
Thanks!
I frequently use acetone precipitation with human or animal samples. The process is similar. I don't know how much that will influence my results compared to yours, but I rarely have issues with it.
That said, have you tried increasing the amount of acetone you use? In my lab we normally use 5x volume and centrifuge for 20 minutes, which works pretty well even for samples with low volumes of protein.
You can also try methanol precipitation (all the steps are the same, you just use cold MeOH instead of cold acetone). I tend to find that acetone works better for my samples, but sometimes different proteins precipitate better with it. It sounds like your protein of interest is too soluble in acetone, so switching to another reagent might be the best choice.