Calculated and apparent molecular weight can differ by 10-20 kDa, therefore careful analysis of obtained band pattern, combined with reliable molecular weight markers is helpful to secure correct interpretation of obtained results. 

There are several reasons which may play a role. To name a few: 

  • N or C-terminal processing, notified in public data bases
  • Posttranslational modifications (PTMs) which increase apparent MW are: phosphorylation glycosylation, Ubiquitination / SUMOylation  (multiple band pattern) NEDDylation 
  • Amino acid composition (+10 kDa)
  • Aggregation or proteolytic cleavage
  • Added epitope tags
  • SDS-PAGE conditions
Recommended: molecular weight markers with IgG binding site, like MagicMarksTM  (Thermo Fisher Scientific) which will appear for each specific MW marker during visualization of Western blot results.

To check if a target protein is subjected to PTMs following approaches can be applied:
  • Phosphatase treatment which will collaqpse phospho-shifted bands
  • PNGase F / Endo H digestion which removes N-linked glycans
  • Deglycosylation enzyme mixes will test glycoproteins
  • Deubiquitinases confirm ubiquitin conjugates
  • Mass spectrometry will provide definitive ID of PTMs




  
Example of difference in calculated and apparent MW of detected protein

Example:
detection of a photosynthetic protein, PsaA, using Agrisera antibody AS06 172
 Technical tips

Technical tips


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