A. Different proteins even with similar molecular weights would exhibit apparent disparity from the resulting SDS PAGE due to the difference in the composition of the protein’s amino acids (e.g. gelatin). The reason for the disparity is due to the amino acids composition that affects the binding of the protein and SDS. Therefore, we can say that protein marker is a handy tool to estimate molecular weight, but there is no absolute molecular weight standard.
B. While running SDS-PAGE, protein mobility can be affected by the composition of the buffer used, gel percentage, the voltage used, running time, as well as if there is a pre-run.
C. Another recommendation for high molecular weight proteins is to prolong the running time to clarify the relative location of bands.