I've successfully transfected series of vectors encoding wide range of protein sizes into HEK293T cells ( they are not cotransfection, instead several different transfections). After harvesting and determining the lysate concentrations, I load the same amount of proteins into SDS-PAGE gel, probe (they all use the same primary and secondary ab), and visualize. My results showed that the biggest protein (~120kda) had the most intense band, while the rest of my samples were decreasing in band intensity as I go from highest to lowest protein sizes (all the way down to ~6kDa).This is something that I've observed while working with DNA.
The size of my observed proteins of interest are as follows:
~120kDa (very strong band)
~10kDa (very faint)
~6kDa (barely visible)
They all need to be on the same gel and the goal of my next repeat experiment is to show all bands in relatively equal intensities. I plan on doing this by lowering the amount of lysate I put in for visualizing the 120kDa protein while increasing the amount of lysate for the rest, by 2x, 4x, 6x, etc. However, these are precious samples so I cannot perform many series using trial and error alone.
Is there a general rule that I can follow? (For example, 10ug of lysate/10kda protein of interest and 1ug of lysate/100kDa protein of interest yields relatively comparable band intensity)
Edited by nocic, 25 November 2013 - 06:32 PM.