1. Why are there multiple bands on my blot?
Several variables may account for this:
a. An excessive amount of lysate loaded onto the gel may cause extra bands. Using decreasing dilutions of lysate will help to determine optimal loading amounts. An increased wash cycle may also alleviate this problem.
b. Lower molecular weight products may be due to protein degradation. We suggest preparing fresh samples by lysing cells/tissues according to our recommended protocol. Compare the signal of your experimental samples with the positive control.
c. Non-specific bands may be due to the secondary antibody. We recommend running a no primary antibody control: verify the appropriate dilution of the secondary detection system, run the Western blot with no primary antibody, and probe only with the secondary detection system.
d. Higher molecular weight products may be a result of post-translational protein modifications, including, but not limited to, glycosylation, myristylation, phosphorylation, and/or ubiquitination.
e. Additionally, higher molecular weight bands may also be a result of protein aggregation that is not resolvable by SDS and boiling.