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, etc. 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 i.HPRD http://www.hprd.org/ ii.ProSite http://www.expasy.org/prosite/ iii.ELM http://elm.eu.org/ 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. 3.Multimerization: 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. 4.Protein charge: The observed size could also potentially be influenced by the protein charge 5.Different species: Different species likely have different protein sequence and PTM, which can lead to a different protein M.W.
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