I'm currently running a western blot with the intent of working out ideal staining conditions. Hence, I have the same 3 proteins repeating on one large gel, and will cut the nitrocellulose and expose them to different Ab concentrations and incubation times. However, I just loaded my gel and noticed that my liver sample is "smiling" within its lane (see attached image). Does anyone know what typically causes this phenomenon?
1) ~50mg skeletal muscle or liver was homogenized with a bead mill. RIPA w/ protease inhibitors was used.
2) Performed a BCA to quantify total protein. Performed in duplicates.
3) Normalized a portion of each protein sample to a concentration of 2ug/uL using the same RIPA w/ protease cocktail.
4) Added 100uL 2x Laemelli to 100uL of each sample. Final concentration protein = 1ug/uL
5) Loaded 20uL (20ug) sample per well. Began run - 150V. Smiling of Liver sample was observed.
I've been using this chamber, gel and buffer system for months - haven't had this issue. However, I previously was loading E.coli lysates that were in 8M Urea. The fact that the adjacent muscle sample is running fine makes me think it's sample specific.
Would protein overloading cause this? I'll look over my math, but thought posting here might be helpful.
Thanks in advance.