Protein ladder size changes down the gel??
 

Hi everyone!

I'm trying to construct a protein ladder for SDS-PAGE. I have conjugated several different molecular weight proteins with dyes and ran them on a 12% gel. There is no problem with the size when I loaded each protein into a separate well, but when I combined all of the proteins (2ul) together into 1 well, the lower molecular weight proteins tend to widen out. The proteins in the ladder are from the same stock as the protein I loaded separately in each well. May someone please explain the reasoning behind this phenomenon or how I can prevent this from happening? I ran the gel at 150V for 10 mins, followed by 200V for 36 mins.

Thank you! Any help would be greatly appreciated!