Limited proteolysis - Partial Proteolytic Digestion
Limited proteolysis is a simple biochemical method which can support information regarding protein structure, conformational changes (1 & 2).
The principle of limited proteolysis is that a protein is incubated with a relatively low concentration of different proteases, which cut at recognition sites throughout the protein, normally at exposed regions such as loops and other flexible regions (3 & 4)
digestion with a protease, samples are analyzed by
The method can be applied to establish the main domain of a protein for crystallization studies, or In situ proteolysis for protein crystallization and structure determination; or to infer the presence of stable sub-domains (1). It is also possible to obtain evidence of binding of one protein to another by running two identical sets of proteolysis experiments, one with the putative target, and one without. If the rate of digestion of the protein is slowed by the addition of the putative target, one can conclude that they interact with each other (1 and 5). Limited Proteolysis can be also be use as a Surface Probe for membrane proteins (7)
- Prepare 1mg/ml of protease in PBS buffer and make 6 dilutions:
2.5µl 2.5µl 2.5µl 2.5µl 2.5µl 2.5µldilute dilute dilute dilute dilute dilute
(1mg/ml) (0.33mg/ml) (0.11mg/ml) (0.0.4mg/ml) (0.012mg/ml) (0.004mg/ml)
- Incubate 20µl of ~0.2mg/ml protein with 5µl of each diluted protease, at 22°C (bath) for 30min.
- Stop incubation of all
proteases by adding 6.5µl of
- Load 15µl on
4.16µl EDTA 500mM
10.4µl PMSF 200mM
 S.J. Hubbard, Biochim. Biophys. Acta 1382 (1998) 191-206.
 Fontana A. et al., Folding Design 2 (1997) R17-R26.
 Quevillon-Cheruel S et al., Methods Mol Biol. 363 (2007) 21-37.
 Moldoveanu T et al , Biochimica et Biophysica Acta 1545 (2001) 245-254
 Dong A. et al., Nature Methods (2007) Vol 12, 1019-1021
 Hargrave P. et al. (1980) Neurochemistry Vol. I, pp. 231-244
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