Amount of protein loaded per well? - (May/26/2009 )

Hi,
how much protein (in micrograms) is common to load to a 3o microliter well. I've seen suggestions from 20 to 200 microliter, but what's the standardamount?

Hi,

Before determining the amount of protein loaded, did u specified the protein concentration by running a protein test such as BCA Assay? The amount of protein load into a well can be determined from the research you are doing. For my case, I'm running a SDS-PAGE with 20 to 60ng of protein with total volume of 20ul (protein sample + dissociation buffer + dH2O).

For a cell extract, I load around 20-30 µg. for a purified protein, I load much less (50 ng, depending on the antibody too)

I've found that for purified protein 5 µg gives a nice band. For cell cultures (e.g. uninduced and induced culture) I calculate the appropriate amount to load based on the OD at 600 nm: If I pellet a 1 ml sample at OD=0.8 I resuspend it in 70 µl buffer/medium/water, mix 15 µl of it with 5 µl 4x loading buffer and load all. For OD=3.5 (overnight induction) I resuspend the 1 ml pellet in 500 µl and do the same. That gives nice comparable samples on my gel (although I don't know if this is the proper way of doing it... ).

yean_ny_nie on May 26 2009, 02:28 PM said: