amount of proteins isolated from cells - (Sep/29/2006 )

Hi,

To prepare proteins for Western Blot, what plate (24-well, 12-well, or 6-well) should I use? How many proteins can be isolated from each type of plates?

Thanks in advance for any suggestion!

This is going to depend on the source of the protein. Are the cells transfected, or is it an endogenous protein to the cells?

For transfected cells, the size of the plate doesn't really matter, since you can only load a small amount of lysate on the gel, so a 24-well plate should be fine. The amount of your protein of interest you can detect will depend on the transfection efficiency and the specificity of your antibody.

For an endogenous protein, it is more tricky if you don't know the expression level of your protein. In that case it would be better to quantitate the amount of total protein in your cell lysate and run set amounts (10 ug, 30 ug, etc) on the gel and just see if you can detect your protein. So the size of the plate will depend on how much cell lysate you need to get that set amount of protein and maybe a 6 well plate would be good so you can get as much as possible.

I don't know how much total protein you can get from each size plate, as that would depend on your cell line, lysis procedure, etc.

~ZP

I'll give my experience


I work with cos-1 cells co-transfecting two plasmids. I plate a 6-well plate at 3-4x10^5, transfect, wait 48 hrs, and then harvest. Typically I get 15-20ug/ul with total volume of 100ul. At end cells are ~75% confluent, if no transfection agent you can expect 1.5x as much.

not sure if this helps.