Protein quantification on spinal cord lysates - reproducibility issues (Jun/08/2007 )

Hello,

I need to do protein quantitation on rat spinal cord lysates. I have used the BCA kit from Pierce on 96 wells plates but I really have reproducibity issues. From one day to the other I can have 10 to 50% difference in my protein concentration.

My lysis buffer (T-PER + protease inhibitors) is compatible with the BCA kit.

I contacted Pierce and they advised me to add 2% SDS in the dilutions of my samples. This is a little more reproducible but still far from perfect.

So either I am really bad at pipeting , either I have a problem with this method.

Is it because spinal cord lysates contain quite a lot of lipids ? Should I use another protein quantitation kit that will be more reproducible ?

Other questions : I need this protein concentration to do ELISA on these samples. Have anyone tried to do an actin ELISA to know if you have the same amount of protein in all your samples ? The same way you do actin WB to compare your signal for other proteins.


Thanks a lot for your replies.


Vero

I don't know if this helps but I have had my most reliable and repeatable
protein conc. results with the Pierce coomasie prtotein reagent
(it's a Bradford assay).
I use new BSA standards and add 1 microliter (or 2 to compare) to ml of the Coomasie reagent
in a disposable cuvette and take the OD at 595 nm.
This is the only method I use to take protein concentrations now and it's great.