animal cell lysis, on plates - (Jan/08/2009 )
Hi, anyone knows a lysis buffer that can protect both protein and dsDNA during/after lysis? and then separate them.
I only have a rough idea of using sucrose and spin a little bit to creat two layers, up for DNA and down for protein.
I need to do this very frequently later on and do not wanna use kits. Can learn nothing from kits.
thanks a lot!
composition of lysis buffer will depend on the type of cells. In our lab we use-
1M Phosphate pH-7+10% deoxycholate+10% NP-40 +1x protease inhibitor cocktail and 4M Nacl. But if you want to do it for separation of proteins on 2D PAGE, then TRIzol lysis would be better.
thanks amj2. at a glance, your buffer is very concentrated, it might be useful for my purpose.
My cells are not characterized yet. Might be a mixture of several cell types, but based morphology, they all looks similar. These cells are stongly attached to the culture dish. I tried to use ripa buffer, but it only solubilize the cytoplasmic protein leaving the cell nucleus on the dish. However, I need both of them for further analysis, enzyme activity assay, etc.