Bradford problem in cell lysates - (Jun/26/2008 )

Hi all

1.-I collected my cells (neurons & astrocytes) in lysis buffer (320 mM sucrose, 1mM DTT, 50 mM Tris, 1 mM EDTA, and minicomplete tablets from Roche with protease inhibitors. pH=7.0). So then I did a Bradford assay and I prepared BSA standards (1 mg/mL the higher and then serial dilutions) in lysis buffer (I suppose standards must be in the same conditions as samples).
But in the zero (just lysis buffer) I got a high absorbance ...could it be due to the protease inhibitors cocktail because these inhibitors are proteinsdi? Do you also prepare your standards in lysis buffer or just distilled water?

2.-And, by the way, how many cells do you need to obtain protein enough to do some WB (10-20 ug/lane)? I use 12-well plates, seed aprox 1.2 x 10e5 cells/cm2, and need one whole plate to get protein enough...one labmate told me she had protein enough with 6 wells of a 12-well plate... Also, does the cell type influence in the protein amount you get?

Thanks a lot

Hi there

For the first questions : I do not make my standards in lysis buffer. And I just found that some inhibitors are proteins, so there is your answer!

2- I cannot really tell you how many cells I use, since I always work with confluent cells. But the one thing I can tell, is that different cell line will give you different amount of proteins. So you have to find how many cells you have to have in order to get enough proteins to work with.

I'm not familiar with 12 wells, but you may want to check your lysis. Such number of cells should give you more proteins. But once again, I never work with 12 wells and neuronal cells. Maybe it is normal.