I'm trying to isolate proteins from sf9's cytosol but I have a problem with cell lysis .
Any idea for lysis buffer or lysis protocol ?
PS: we do NOT want to centrifuge cells then lyse them .. we harvest them using crossflow filtration and then add lysis buffer to retentate ..
in the other words, we want to keep cells within crossflow and lyse them to proceed next step - Clarification -
Thanks.
If you cannot use sonication since you want to keep the cells within the crossflow, then you can use a chemical lysis method - you could use I-PER (Insect protein extraction reagent, thermo-fisher) along with some Benzonase nuclease to deal with viscosity from the dna of lysed cells.
Use a mild detergent. The Nonidet P-40 (don't confuse it with NP-40, those are different detergents) is very mild, so mild it only lyses the plasma membrane, leaving the nuclear membrane intact, so no DNA problems. Try 0.1 to 0.5% of Nonidet P-40, see what works best for you. The NP-40 lyses everything, it's confusing, I know! You can also try CHAPSO, but this, even though is mild, lyses everything too.