Hi,
I have been working with C.elegans for 9 months now. about 6 months ago I made a topic in here asking for help on breaking the worms efficiently for large scale purification of proteins.
As I got no answers I just went for it and I have now a pretty efficient protocol.
Basically I employ 3 lysis techniques in a row: Mortar and pestle, Tissue Grinder and Nitrogen bomb.
NB: Every step is performed in a 4C cold room, this is important for my protein, but may not be for important for you. but hey, it normally never hurts to keep proteins cool!
I start by harvesting my worms by standard protocol, except I have maybe 10-15 ml of hard pelleted worms when I am done (large scale) so scale the protocol to your needs.
Then I freeze the worms directly in a cryo-cooled mortar drop by drop. Im not using anything fancy, just a cheap standard porcelain mortar and pestle. The trick on how not
to break the mortar, is simply having liquid nitrogen on both sides of the mortar. I have the mortar standing in a container suitable for liquid nitrogen and then I pour
nitrogen directly into the mortar before starting and top it off when ever needed. This way the mortar have lasted a long time compared to mortars in other labs at our department where they only have nitrogen inside the mortar.
Anyway!
I proceed to smash/grind the worms with the pestle until I have a fine powder (the finer the powder the better). It takes some time when you have the amount of worms I have, but it is really worth it in the end! Then I resuspend the powder in lysis buffer (1.5 ml per gram powder, the composition of the lysis buffer differs with the target protein etc).
Dont worry if the powder freezes when you add the lysis buffer, the powder is really cold so it is normal. Actually its better than waiting for the powder to warm up.
The sample is then put through a tissue grinder like this one. I move the piston up and down 8 times per load (I have more sample than can fit into the grinder). Mind you the first two “grinds” can requirer a lot of force sometimes (hence the fine powder in the beginning)!
After the tissue grinder I directly pour the sample into a pre chilled (I have in our cold room) nitrogen bomb together with a magnet (for stirring) and place it on a standard magnetic stirrer (medium to high speed needed). The chamber is sealed and charged it with nitrogen gas until a pressure 1200 of psi is reached.
Dont worry you can very easily tell if the chamber is not tight, as the gas will diffuse out rapidly immediately upon charging the chamber.
I leave the gas to incubate for a hour and a half (90 min). The longer time and the higher the pressure you leave the sample in the more nitrogen the cells will take up and the better the cell disruption will work when releasing the pressure.
you can play around with the pressure and incubation time it should be possible to even break mitochondria with the nitrogen bomb, if you need that.
I release the pressure while collecting the foam directly into a 50ml tube, dont worry this is the good type of foam, nitrogen foam not oxygen foam, so your proteins are fine. A word of caution the last part of the foam will leave the chamber quite fast and suddenly making the rubber tube go flying if not secured (I just hold in my hands) and you will be sprayed in C.elegans foam (dont that, never again).
The foam can easily be spun down to liquid(10 min at 4000 rpm, standard).
At this point I have no worms left at all, not even partly open worms or cuticles!!! A quick inspecting of the sample under a dissecting microscope can easily tell you how you did!
it is a pretty harsh lysis technique but it works perfect for me and I have achieved by fare the highest amounts of protein this way.
I truly hope that someone can use this protocol, but unfortunately it seems I am one of the rare persons that purify proteins with C.elegans as source, which I dont get because it is easy to scale the production up!
Anyway feel free to ask if you have any questions comments or improvements.
/ruki