Why is the observed Western Blot band size different from predicted size?
The predicted M.W. is based on protein sequence analysis; however, some factors might lead to an observed band size that is different from the predicted size. The reasons might include:
1.Post-translational modification (PTM):
a. Some post-translational modifications might lead to increased protein size, including
phosphorylation, acetylation, methylation, glycosylation, sumoylation, ubiquitination,
b. Some post-translational modifications might lead to decreased protein size including
phosphatidylethanolamine conjunction (e.g. LC3-II)
c. Some proteins may be cleaved to form an active or mature form; this process will
lead to a decreased protein size (e.g. Notch activation, Caspase activation, etc.)
d. Some websites provide useful PTM information
iv.CBS data sets http://www.cbs.dtu.dk/databases/
v.CBS prediction Servers http://www.cbs.dtu.dk/services/
2.mRNA splice variants (Isoforms):
Through alternative splicing, one gene can generate different proteins with different M.W. Regulation of alternative splicing depends upon cell type, conditions, etc.
Some proteins could form dimers or multimers, increasing the M.W. This phenomenon usually can be found in reducing gel condition; however, strong interactions may still be seen with higher molecular weight proteins even in denaturing gel.
The observed size could also potentially be influenced by the protein charge
Different species likely have different protein sequence and PTM, which can lead to a different protein M.W.