Protein amount to load for publication figure? - (May/10/2013 )
I need a figure of a SDS-PAGE gel with a couple different purified proteins. I tend to overload gels because I am just checking quality and size and just load "a few microliters". How much (in ug) protein should be loaded per well? Each sample is pure (only 1 protein) and I am looking to get a normal size band that is more intense than the ladder, but not overwhelming.
For standard Coomassie staining, I generally load about 2ug of a singular purified protein per well. You should get a crisp dark band. In my experience, if you go much over 5ug your band will start to become more blob and less band.
how are you going to stain the gel?
if you use coomassie then you want 1-5ug (depends on the size of the protein, width of the well, thickness of the gel, etc).
if you stain with silver then no more than ~100ng.
If you have enough of your purified protein, can't you just run several lanes each with a different amount of protein and then pick the amount that gives you the best image?
Thanks for the suggestions. I will be coomassie staining on a 1mm gel. The sizes are 20 & 70 KDa. I will try the 2ug and see how that goes