Cell Lysates for western - (Apr/04/2006 )

Hi everybody!

I was wondering if someone can tell in which volume I have to ressupend my lysate after boiling 10 6 cells for western. Because I use to extract nuclear extract for my western and this is the first time I will try with cell lysates.
Thanks in advance

I can depend on your type of cell. I keep my volume for resuspension the same every time; 200 µL of my home-made single-detergent lysis buffer. Then after doing a Bradford I just alter the volume I add to my samples.

30% of a 175 cm^2 flask will give you 200 µL at 15 mg/mL (that's 3 mg total protein). Until you've performed Bradford you will never be too sure.

a good ratio between the quantity of cells and quantity of detergent is very important for protein extraction. This depends on your cells (on the quantity of protein to extract from your cells).
You can go from 106 cells to 5.106 cells in 100 uL of lysis buffer.
Just to give you an idea, I solubilized 106 cells in 100 uL, and diluted three times a little aliquote to perform Bradford protein concentration determination.

you can concontrate your proteine by acetone precepitation.
add 1V of acetone chech breafly and centrifuge 15 min at 1200-1300rpm
disssolve the pellet in sterile water and determine the protein yield by Bradford reagent
goooooooooooood luck

Thank you very much for all your answers