Cell lysis method for running cell lysate on Western blot - (Apr/24/2008 )

Hi,

I am trying to express a His-tag protein in 293-T cells, which has an IL-2ss for secretion. I tried to isolate the protein from the culture supernatant using Dynabeads TALON, but i didn't get any protein. I would like to see if the protein is expressed but not secreted. So i thought of lysing my transfected cells and running the cell lysate on a Western blot. Can anybody suggest an appropriate menthod for preparing a cell lysate of 293-T for running on a Western blot please?

Thank you for reading.

I would lyse cell pellet in loading buffer, heat 5 minutes, and load hot (because it will be goopy). Someone else will have to tell you how many cells/well to load, it has been a while since I've done it.
D

Usually just use Laemli buffer and heating will lyse the cells unless u really want to use sonication to lyse it completely.

Thanks alot for your replies.

thanks for the replies. i was looking up for the same!