I never used this. To make protein lysates from tissues, I do old school sonication. 100% intensity, 5s pulses all on ice. It works for everything. For when i need to isolate synaptosomes or to do protein-protein interactions, i use a dounce homogenizer. good luck!
We use the Precellys for RNA and protein extractions and it seems to work well in our hands. For human or mouse DRG tissues, I use the Precellys Soft Tissue Homogenizing beads but for other tissue types such as the sciatic I usually opt for the metallic beads. As for the buffers, I typically use RIPA, or the Tissue Protein Extraction Reagent spiked with protease and phosphatase inhibitor cocktails.
Typical Precellys procedures is as follows:
RPM: Highest speed setting.
We have the older version of the Precellys homogenizer with just low, medium, or high settings.
You can increase the number of cycles as needed.
Cycle Duration: 30s – 45s
Delay between cycles: 30s
And like @AubinMoutal, we also use old-school sonication, and it works pretty well too! Hope this helps.
Depending on the tissue quantities, my volumes can range from 100-450ul. Since it looks like you are homogenizing only 3 mouse DRGs (been there!), I would also suggest decreasing the lysis volume to maybe 100ul or 150ul. Note: 100uL lysis buffer may be a little tricky to homogenize. To help with this, I use the Precellys 200ul tubes with a screw cap and I find it really helps.
And yes, I put them on ice in between cycles. Our homogenizer is also kept in the cold room.
Thanks! Yup I’m using between 100-200 ul for that quantity of mDRG.
I use the CK14 tubes in the 0.5 mL volume.
I will keep messing around with volumes and cycles until we get it right. I want this to work. I like the throughput and the ease of just using beads, but we need to improve extraction so that’s comparable with hand grinding.