protein extraction from mammalian cell line - preferred protocol? - (Jul/31/2008 )

Hello,

I'm new to proteins. I am looking for a good protocol for protein extraction from a mammalian (mouse) cell line (3T3-L1). I don't have much experience in the field, so even the most basic or detailed protocols would be helpful (i.e. how many cells do I need to have to start, etc). I am looking to use the protein for Westerns and to coat wells for ELISA.

Thanks.

My cell line!! ^^

First of all, for the number of cells, it all depends on what you want to test on these cells. Since they a preadipocyte, I suppose you want to differentiate them into adipocyte. You surely know that in order to differentiate, these cells need to be brought to confluence. So the number of cells is pretty variable.

As for the protein extraction, I usually lyse cells in slightly modified RIPA buffer. For a 60 mm, I lyse cells in 500 µl, or 1 ml for a 100 mm. Depending on the state of differentiation, you will get from 1,6 to 4,1 µg of proteins by µl.

So, for the protocol.

On the day of the extraction, scrape the cells in the petri dish (you can wash cells first with PBS if you want to), leaving the media in the dish. Transfert the cell suspension into a falcon, and spin down the cells at 1000g.

Then remove the supernatant carefully. You have to be pretty careful with 3T3-L1, since differentiated cells tend form a slurpy media. Removing the media with a suction pump will inevitably result in losing your pellet in the pump. Trust me on that one

Add the chilled RIPA buffer with protease inhibitors in the tube, and lyse cells mechanically by passing the pellet 5 times into a 23g needle with a syringe. Place the mixture into an eppendorf and rock at 4 c for 1 hour.

Spin 5 min at 13000g and transfert the supernatant into a new eppendorf. And you're done.

Hope this helps!

My cell line!! ^^

First of all, for the number of cells, it all depends on what you want to test on these cells. Since they a preadipocyte, I suppose you want to differentiate them into adipocyte. You surely know that in order to differentiate, these cells need to be brought to confluence. So the number of cells is pretty variable.

As for the protein extraction, I usually lyse cells in slightly modified RIPA buffer. For a 60 mm, I lyse cells in 500 µl, or 1 ml for a 100 mm. Depending on the state of differentiation, you will get from 1,6 to 4,1 µg of proteins by µl.

So, for the protocol.

On the day of the extraction, scrape the cells in the petri dish (you can wash cells first with PBS if you want to), leaving the media in the dish. Transfert the cell suspension into a falcon, and spin down the cells at 1000g.

Then remove the supernatant carefully. You have to be pretty careful with 3T3-L1, since differentiated cells tend form a slurpy media. Removing the media with a suction pump will inevitably result in losing your pellet in the pump. Trust me on that one

Add the chilled RIPA buffer with protease inhibitors in the tube, and lyse cells mechanically by passing the pellet 5 times into a 23g needle with a syringe. Place the mixture into an eppendorf and rock at 4 c for 1 hour.

Spin 5 min at 13000g and transfert the supernatant into a new eppendorf. And you're done.

Hope this helps!