Biology Protocols

  • Increase font size
  • Default font size
  • Decrease font size

Limited Proteolysis of proteins

E-mail Print PDF

Limited proteolysis is a relatively simple biochemical technique that can provide important information on the structure and dynamics of proteins.

The principle of limited proteolysis is that a protein is incubated with a relatively prolific protease, such as trypsin, which cuts at recognition sites throughout the protein, normally at exposed regions such as loops and other flexible regions.

Following digestion with a protease, samples are analysed by SDS-PAGE to identify cleavage products. The appearance of lower molecular weight bands denotes digestion of the full length protein, and intensity and appearance/disappearance of bands can be closely monitored and quantified. Proteolytic cleavage product sequences can be predicted based on molecular weight assignment.

This allows the prediction of structure based on the amino acid sequence of the protein to be studied.

For example:

Trypsin predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine, except when either is followed by proline. Therefore if a short peptide has a sequence


Cleavage would be expected to occur and produce peptides:


If, however, the following cleavage products were observed:


It could be assumed that lysine highlighted in red was embedded within the molecule and therefore inaccessible to tryptic digest and the other products had no specific structure.

Further incubation with trypsin would be expected to yield the full complement of cleavage products.

Add this to your website