Protease and phosphatase inhibitors are essential components of most cell lysis and protein extraction procedures. These inhibitors block or inactivate endogenous proteolytic and phospholytic enzymes that are released from subcellular compartments during cell lysis and would otherwise degrade proteins of interest and their activation states.

What are proteases (proteolytic enzymes) and phosphatases?

Proteases and phosphatases are important enzymes in a variety of biochemical pathways in living cells. Proteases are required for many cellular functions, including cellular repair and the digestion of extracellular material. Phosphatases play a key role in regulating signal transduction events in eukaryotic cells. Protein kinases transfer a phosphate from ATP to a serine, threonine, or tyrosine residue in a protein; phosphatases remove the phosphoryl group. Phosphorylation is the most common post-translational modification on proteins, with approximately 80% occurring on serine, 20% on threonine, and 0.1 to 1% on tyrosine residues.

All living organisms contain proteolytic enzymes (proteases and peptidases). In whole cells, protease and phosphatase activities are tightly regulated by compartmentalization or inhibitors to prevent indiscriminate damage to cellular proteins and to maintain proper function of signaling pathways. Cell lysis disturbs the carefully controlled cellular environment, allowing proteases and phosphatases to become unregulated. The usual consequence of this unregulated state is reduced recovery of total protein and biologically meaningless representation of protein activities (i.e., phosphorylation status).

Graphical depiction of cellular compartments that control protease and phosphatase activities, but cellular disruption leads to all proteins being accessible for degradation
Protein degradation during cell lysis. Many of the cellular proteins are kept separate from proteolytic enzymes. Disruption of cellular and tissue architecture during protein extraction distorts the in vivo state by making all proteins potentially accessible for degradation or modification by endogenous proteases and phosphatases. The resulting unregulated proteolytic activity can reduce protein yield and function. Protease and phosphatase inhibitors can be added to the lysis reagents in order to prevent degradation of extracted proteins, and to obtain the best possible protein yield and activity following cell lysis.


Inhibition of protease and phosphatase activity

Protease inhibitors are biological or chemical compounds that function by reversibly or irreversibly binding to the protease. Most known proteases belong to one of four evolutionarily distinct enzyme families based on the functional groups involved in cleavage of the peptide bond. Known phosphatases are specific for cleavage of either serine-threonine or tyrosine phosphate groups. Thus, while numerous compounds have been identified and used to inactivate or block these enzymes, no single chemical is effective for all types of proteases and phosphatases (see tables below). Rather, a mixture or inhibitor cocktail of several different inhibitor compounds are used to ensure that protein extracts do not degrade before analysis for targets of interest. Proteases inhibitors are nearly always needed, while phosphatase inhibitors are required only when phosphorylation states (activation states) are being investigated. Research experiments may necessitate the use of single inhibitors or customized mixtures, but most protein work is best served by using a suitable protease inhibitor cocktail.

Commonly used protease inhibitors

InhibitorMW (kDa)Target classTypeSolubility (solvent)Typical working (1X) conc.
AEBSF239.5Serine proteasesIrreversible200 mg/mL (H2O)0.2 to 1.0 mM
Aprotinin6511.5Serine proteasesReversible10 mg/mL (H2O)100 to 200 nM
Bestatin308.4Amino-peptidasesReversible5 mg/mL (MeOH)1 to 10 µM
E-64357.4Cysteine proteasesIrreversible20 mg/mL (1:1 EtOH:H2O)1 to 20 µM
EDTA372.2Metalloproteases (chelates cations)Reversible10 g/100 mL (H2O)2 to 10 mM
Leupeptin475.6Serine and cysteine proteasesReversible1 mg/mL (H2O)10 to 100 µM
Pepstatin A685.9Aspartic acid proteasesReversible1 mg/mL (MeOH)1 to 20 µM
PMSF174.2Serine proteasesReversible18 mg/mL (MeOH)0.1 to 1.0 mM

Commonly used phosphatase inhibitors

InhibitorMW (kDa)Target classTypeSolubility (solvent)Typical working (1X) conc.
Sodium fluoride42.0Ser/Thr and acidic phosphatasesIrreversible40 mg/mL (H2O)1 to 20 mM
Sodium orthovanadate183.9Tyr and alkaline phosphatasesIrreversible20 mg/mL (H2O)1 to 100 mM
beta-Glycerophosphate (Disodium salt)216.0Ser/Thr phosphatasesReversible10 mg/mL (H2O)1 to 100 mM
Sodium pyrophosphate221.9Ser/Thr phosphatasesIrreversible65 mg/mL (H2O)1 to 100 mM
Prevention of protein degradation by proteases after cell lysis using various protease inhibitor cocktails

Comparison of commercially available protease inhibitor cocktails and tablets. Pancreatic extract (50 μL, 1 μg/μL protein) or trypsin (25 μL, 0.1 units/μL) was incubated with a quenched-fluorescent, protease-cleavable substrate for cysteine (A) or serine proteases (B) in the presence or absence of commercially available protease inhibitors with EDTA-containing (blue) or EDTA-free (purple) formulations. Reactions were incubated for 2 hours at 37°C and the fluorescence determined at indicated detecting emissions. The percent protease inhibition is shown for each protease inhibitor formulation.

Recommended reading

  1. Walker JM (2009) The Protein Protocols Handbook. Third Edition. New York (NY): Springer-Verlag New York, LLC.

Watch this video to learn how to use Thermo Scientific Pierce phosphatase and protease inhibitor tablets