Hello,

I work with HaCaT cells, and until now there hasn’t been any issues. But recently I’ve had a problem when lysing the cells. Upon the addition of lysis buffer, the cell “suspension” turns into jelly and become gelatinous. When I try to aspirate with a pipette, the suspension is very sticky and acts much like snot. There is also no cell pellet when I try to spin it down.

Usually I use a ready made RIPA buffer with the addition of protease/phosphatase inhibitor, but I get the same results if I make it myself (fx. A NP-40 lysis buffer). Through some experimentation I have found that it is the addition of detergent that causes this. I’ve tried to replace detergents (SDS, Triton, Tween) and varying the concentration (1-0.01%) but no matter what the suspension turn into jelly - if not right away then at some later point.

I’ve read that RIPA buffer can turn gelatinous if heated, but the buffer itself in bottle looks fine and works fine with tissue samples. Everything is also kept on ice, so I don’t believe that to be the issue.

The result is the same if I resort to add the buffer directly onto the plastic ware and use a cell scraper to get the cells off.

I have thawed a new batch of cells, but the result is still the same.

Currently I resuspend in media with added inhibitor cocktail and then do a mechanical lysis. But at some point I would like to look at membrane proteins and would then need a proper lysis of the cells.

Does anyone know what could be going on? I would appreciate any advice to solve this issue.

Thank you.