Hey all, was hoping to find what people’s favorite way to make lysates for western blots from samples that include embryos. I had been using the approach from Michael Koelle’s website (freeze-thaw 3x in a buffer lacking glycerol then boil in sample buffer) but was wondering if there were more effective ways. I’ve seen reports of boiling, freezing, and then re-boiling, or sonication. Thanks!
Hey Jordan,
My favorite way to make any worm extract is to resuspend the packed worm pellet in 2X RIPA at an equal volume to the pellet. If its a transcription factor that you think might stick to the DNA, then use 450 mM NaCl rather then the standard 150 mM. We add proteosome inhibitor, protease inhibitor, DTT to every extract. I aim for around 100 ul, so there is enough for our sonicator. After sonication, clear with a spin, check protein, add loading buffer and boil. I’ve used this for all sorts of different proteins. If your embryo pellet is small, you may not need to use the 2X RIPA, just add 1X to the pellet at an amount you can sonicate.
Hope this helps,
Amy
Thanks Amy, that’s super helpful! What sonication parameters do you usually use (time, % power, 1 vs numerous cycles)?
cheers,
Jordan
We use 20% amplitude, then do 10 seconds on, 20 seconds off. I do this 5 times, on ice. With the adults, you can see if there are some that aren’t lysed and if that is the case, we do a few more cycles. For the embryos, you could try 3X, check on the dissecting scope and then do 5 if they weren’t lysed.
I’ve done this with multiple different micro tip sonicators, so it should work on your standard model.
Hope this helps!
Amy