The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker  Tel: 972-2-6586920 

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)

Following digestion with a protease, samples are analyzed 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 (3). Variables that can be used for results optimization: type of protease, protease dilution, temperature or time of incubation.

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)


1) Trypsin

2) Trombin

3) Subtilisyn

4) Chymotrypsin

5) Others

- Prepare 1mg/ml of protease in PBS buffer and make 6 dilutions:

               2.5µl                2.5µl                 2.5µl               2.5µl                  2.5µl                2.5µl

              dilute                dilute                dilute               dilute                 dilute                dilute
7.5µl                  5µl                  5µl                    5µl                     5µl                    5µl                   Out

(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 SDS stop solution sample buffer X 5 (with 5.2mM PMSF & 5.2mM EDTA), boil at 100°C 3 to 5 min and load on gel immediately. 

- Load 15µl on PAGE-SDS gel of untreated proteins and the different treated proteins.


Stop solution:

4.16µl EDTA 500mM

10.4µl PMSF 200mM

SDS-Sample buffer X 5 up to 400µl


[1] S.J. Hubbard, Biochim. Biophys. Acta 1382 (1998) 191-206.

[2] Fontana A. et al., Folding Design 2 (1997) R17-R26.

[3] Quevillon-Cheruel S et al., Methods Mol Biol. 363 (2007) 21-37.

[4] Moldoveanu T et al , Biochimica et Biophysica Acta 1545 (2001) 245-254

[5] Chazin Lab Protocol for Limited Proteolysis:

[6] Dong A. et al., Nature Methods (2007) Vol 12, 1019-1021

[7] Hargrave P. et al. (1980) Neurochemistry Vol. I, pp. 231-244



Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,
The Hebrew University of Jerusalem  Tel: 972-2-6586920  

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