lysing cells on ice - (Mar/11/2010 )

Hi,


I'm new to working with cell cultures and although i'm not experiencing problems with my experiment, I can't seem to figure out why cells need to be lysed on ice with icecold lysis buffer(Tris,EDTA,Triton x-100, NaCl and protease inhibitors). I'm probably missing the obvious, answers are more than welcome!

Dfr on Mar 11 2010, 03:43 PM said:

Hi Dfr,

Once you lyse your cells, the system is disrupted that produced hopefully happy soluble protein. Proteases can attack your proteins and cause degradation. Warm temps can cause your protein to precipitate. Protease inhibitors are not a cure all for this and don't inhibit all the proteases from your cells. Think of any enzyme reaction at different temperatures. Warmer temps increase activity rate.
So freezing your cell pellet with cells intact, then thawing and keeping everything that comes in contact with your cell lysate ice cold, you are buying some time to separate your protiein from harmful proteases before they do much damage.

Only thaw the amount of cell pellets that you will be able to run through your first purification step the same day. Leaving cell lysate overnight even at cold temps will allow the proteases that are present to chew away at your proteins. You want to get your protein out of the unfavorable environment and into buffers that will help stabilize it quickly.

Thanks for the help !