protein extraction protocol? - (Sep/04/2008 )

hey all,

i need to extract total protein from a mammalian cell line (3t3-l1) cells to run a western blot. i was wondering if anyone has a good protocol for protein extraction that uses protease inhibitors. also, how many cells (approximately) do i need to get a sufficient protein yield? i transfected a gene-gfp fusion construct into these cells and they are only weekly glowing under the scope so i want to confirm the presence of the construct with a western blot.

I will refer you to a previous post of yours, where I answered these questions

http://www.protocol-online.org/forums/inde...mp;#entry145773

I will also add that 3T3-L1 are really difficult to transfect. People generally use viruses (retro or lenti) to deliver material to these cells.

We cannot remember everything

Hope this helps!