Description
GoldBio’s Leupeptin Hemisulfate is a molecular biology grade, reversible protease inhibitor used to limit protein degradation during cell and tissue extraction and to investigate protease-dependent processes.
Leupeptin inhibits several serine and cysteine endopeptidases, including trypsin, plasmin, kallikrein, papain, calpain, and cathepsin B. This inhibition profile makes leupeptin useful as an individual inhibitor or as a component of broader protease inhibitor cocktails when you need protection against multiple trypsin-like and cysteine proteases.
Leupeptin is also widely used as an experimental lysosomal protease inhibitor. By slowing lysosomal protein degradation, it can be used to examine protein turnover and quantify macroautophagic flux through accumulation of lysosome-sensitive markers such as LC3-II.
Working Concentration
The typical working concentration ranges from 10 to 100 µM.
Stock Solution Preparation (10mM Leupeptin Stock Solution)
- Weigh 5 mg of Leupeptin Hemisulfate.
- Pipet 1.05 ml of pure sterile H2O.
- Mix until completely dissolved.
- Use immediately or store at -20°C for 1 month.
Note: Leupeptin is effective at a concentration of 10-100μM. Dilute the stock solution 1:100 for a 100μM working solution. Leupeptin Hemisulfate is also soluble in DMF, DMSO, and ethanol.
Common Applications
(Click each for more information)
Protection of Proteins During Cell and Tissue Lysis
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Purpose: To reduce proteolytic degradation of target proteins during sample extraction and preparation.
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How It Works: Leupeptin reversibly inhibits multiple serine and cysteine proteases that can be released or activated during cell disruption. Adding it to cold lysis or extraction buffers helps preserve susceptible proteins before downstream analysis.
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Applications: Preparation of cell and tissue lysates, immunoblotting, immunoprecipitation, protein quantification, and protein purification.
Libby, P., & Goldberg, A. L. (1978). Leupeptin, a protease inhibitor, decreases protein degradation in normal and diseased muscles. Science, 199(4328), 534–536. https://doi.org/10.1126/science.622552
Measurement of Macroautophagic Flux
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Purpose: To estimate the rate at which autophagic material is delivered to and degraded within lysosomes.
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How It Works: Leupeptin inhibits lysosomal proteases and slows degradation of autophagic cargo. Comparing accumulation of markers such as LC3-II in the presence and absence of leupeptin provides a chemical-clamp approach for measuring autophagic flux rather than relying only on steady-state autophagosome levels.
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Applications: In vivo and ex vivo autophagy studies, LC3 turnover assays, lysosomal degradation studies, and analysis of changes in macroautophagic activity.
Haspel, J., Shaik, R. S., Ifedigbo, E., Nakahira, K., Dolinay, T., Englert, J. A., & Choi, A. M. K. (2011). Characterization of macroautophagic flux in vivo using a leupeptin-based assay. Autophagy, 7(6), 629–642. https://doi.org/10.4161/auto.7.6.15100
Lysosomal Protein Degradation Studies
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Purpose: To distinguish lysosome-dependent protein degradation from other intracellular protein turnover pathways.
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How It Works: By inhibiting lysosomal cysteine proteases, leupeptin suppresses a substantial portion of lysosomal proteolysis. Changes in protein degradation or substrate accumulation after treatment can therefore be used to investigate the contribution of lysosomes to intracellular protein turnover.
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Applications: Protein turnover experiments, lysosome function studies, degradation-pathway comparisons, and analysis of lysosomal processing of cellular proteins.
Seglen, P. O., Grinde, B., & Solheim, A. E. (1980). Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. European Journal of Biochemistry, 105(2), 215–220. https://doi.org/10.1111/j.1432-1033.1980.tb04495.x
Calpain Inhibition and Calpain-Dependent Proteolysis Research
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Purpose: To suppress calpain activity and examine the contribution of calcium-dependent proteolysis to cellular or biochemical processes.
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How It Works: Leupeptin inhibits calpain by binding reversibly to its proteolytic active site. Researchers can compare reactions or samples with and without leupeptin to evaluate whether observed protein cleavage or structural changes depend on calpain activity.
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Applications: Calpain activity studies, proteolysis assays, cytoskeletal protein research, and biochemical studies of calcium-dependent protease function.
Saito, K. I., & Nixon, R. A. (1993). Specificity of calcium-activated neutral proteinase (CANP) inhibitors for human μCANP and mCANP. Neurochemical Research, 18(2), 231–233.
Cathepsin B and Cysteine Protease Inhibition Studies
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Purpose: To investigate cathepsin B activity and the kinetics or biological consequences of cysteine protease inhibition.
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How It Works: Leupeptin forms a tight, reversible complex with cathepsin B. Its concentration-dependent inhibition allows researchers to suppress cathepsin B activity and examine enzyme kinetics, substrate processing, and protease-dependent degradation.
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Applications: Cathepsin B enzyme assays, cysteine protease characterization, inhibitor studies, and lysosomal protease research.
Baici, A., & Gyger-Marazzi, M. (1982). The slow, tight-binding inhibition of cathepsin B by leupeptin: A hysteretic effect. European Journal of Biochemistry, 129(1), 33–41. https://doi.org/10.1111/j.1432-1033.1982.tb07017.x
Key Benefits
Broad Serine and Cysteine Protease Coverage: Inhibits several commonly encountered proteases, including trypsin, plasmin, kallikrein, papain, calpain, and cathepsin B.
Reversible Inhibition: Allows strong protease suppression without relying exclusively on irreversible active-site modification.
Useful for Protein Protection: Helps limit proteolytic loss of susceptible proteins during cell or tissue extraction and sample preparation.
Established Lysosomal Research Tool: Supports studies of lysosomal protein degradation and macroautophagic flux by slowing proteolysis within lysosomes.
Flexible Use: Can be used alone for targeted inhibition or combined with complementary inhibitors in protease inhibitor cocktails.
Molecular Biology Grade: Supplied by GoldBio as molecular biology grade material for research applications.
Storage/Handling
Store at -20°C.
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