There are a few rules about loading protein samples on SDS-PAGE gels.
The first is that if you overload the gel with excess protein, you risk streaking of blots. Also if you expect to see differences in the lanes, this may be a problem as the blots easy saturate upon ECL development with excess protein (large bands very dark for all).
SO here are some loading rules:
1) The amount of protein you load depends on the well size and gel thickness. It has been noted that if you load 10 µl of a 2 mg/ml final concentration of denatured protein per sample well, you get better gel usually.
2) In general, the amount of protein sample you should load onto your SDS-PAGE gel should range from 0.5–4.0 µg for purified (recombinant) samples to about 40–60 µg for crude samples (this includes Coomassie blue staining)
Silver staining methods are about 100-fold more sensitiveand thus require less protein per sample.
3) Working concentrations of proteins should all be equal throughout the gel to prevent irregularities in running (due to differences in the gel salt etc) and most importantly to be able to compare different treatments / conditions (different lanes).
If anyone has more information or tips, please contribute them here!