As mentioned above, I am having trouble with my subcellular fractionation of 4T1 cells.

For my cytoplasmic lysis buffer, I am using 1% TritonX-100 Lysis buffer (20mM HEPES, 50mM NaCl, 1.5mM MgCl2, 2mM EDTA, 1% TritonX-100) with the following inhibitors: 1mM DTT, 5mM NaF, 1mM PMSF, 4mM Sodium Orthovanadate, and 2% PICm (protease inhibitor cocktail for mammalian cell lysate).

For my nuclear lysis buffer, I am using the exact same concentrations, albeit with addition of 0.1% SDS, and with the aid of sonication, nuclear lysis is easily performed.

The problem is that when I add the cytoplasmic lysis buffer to my pelleted cells (flicked a few times before addition of buffer), the cells seem to be unable to withstand the lysis and the nucleus of the cells lyse immediately upon addition of the CLB, when it should not be lysing in CLB. I can tell that the nucleus has lysed due to the lack of observable nucleus under microscope (1 ul lysed cells: 10 ul tryptan blue) and the presence of a sticky occurence in the cytoplasmic lysate.

The odd thing is that if I remove sodium orthovanadate and NaF from the inhibitor cocktail, lysis is effective and I get extremely clean nuclear and cytosolic fractions. However, upon addition of sodium orthovanadate and NaF, my cell's nuclei lyse instantly upon addition of the CLB.

I suspect that the concentration of TritonX may be too high, but I am not very sure why the addition of sodium orthovanadate and NaF to the CLB may cause instant lysis of my cell's nuclei. I am currently looking to decrease the concentration of TritonX to 0.1%, and lowering NaCl to perhaps 10-50 mM. Would this be a good way to troubleshoot?