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 cells lysis and would otherwise degrade proteins of interest and their activation states.

Proteases 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).

Protein degradation. 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.

Cell and Protein Isolation Technical Handbook

Learn how to optimize protein extraction from cells and tissue for better yield and improved downstream compatibility using Thermo Scientific Pierce Protein Biology Products.

  • Extract total protein quickly and efficiently
  • Obtain subcellular fractions with minimal cross- contamination
  • Enhance lysis and immunoassay techniques with highly purified, prediluted detergents
  • Remove interfering detergents rapidly and effectively

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 table).

Rather, most researchers prepare or use a mixture or "cocktail" of several different inhibitor compounds 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. Particular 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 (see additional discussion below table).

Commonly used protease and phosphatase inhibitors.

Inhibitor MW Target class Type Solubility (solvent) Typical working (1X) conc.
Sodium Fluoride 42.0 Ser/Thr and acidic
Irreversible 40mg/mL 
1 to 20mM
183.9 Tyr and alkaline
Irreversible 20mg/mL 
1 to 100mM
(disodium salt)
216.0 Ser/Thr
Reversible 10mg/mL 
1 to 100mM
221.9 Ser/Thr
Irreversible 65mg/mL 
1 to 100mM
AEBSF•HCl 239.5 Serine proteases Irreversible 200mg/mL 
0.2 to 1.0mM
Aprotinin 6511.5 Serine proteases Reversible 10mg/mL 
100 to 200nM
Bestatin 308.4 Amino-peptidases Reversible 5mg/mL 
1 to 10µM
E-64 357.4 Cysteine proteases Irreversible 20mg/mL
(1:1 EtOH:H2O)
1 to 20µM
EDTA 372.2 Metalloproteases
(chelates cations)
Reversible 10g/100mL 
2 to 10mM
Leupeptin 475.6 Serine and cysteine proteases Reversible 1mg/mL 
10 to 100µM
Pepstatin A 685.9 Aspartic acid proteases Reversible 1mg/mL 
1 to 20µM
PMSF 174.2 Serine proteases Reversible 18mg/mL 
0.1 to 1.0mM

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.

Inhibitor cocktails and tablets

We offer a variety of both individual protease inhibitors and ready-to-use, broad-spectrum protease and phosphatase inhibitor cocktails. The inhibitor cocktails are available as both 100X cocktail solutions (i.e., Thermo Scientific Halt cocktails) and quick-dissolving tablets (Thermo Scientific Pierce tablets) to accommodate specific and general needs in cell lysis and protein extraction methods. These inhibitors are suitable for the protection of proteins during their extraction from cultured cells, animal tissues, plant tissues, yeast or bacteria.

Our Thermo Scientific protease inhibitor cocktails and tablets target serine-, cysteine- and aspartic acid proteases and aminopeptidases. Metalloproteases are inhibited by the optional addition of EDTA (available in a separate vial in the cocktail format but included in the tablet format). Our Thermo Scientific phosphatase inhibitor cocktails and tablets contain chemical compounds that target serine/threonine and tyrosine phosphatases.

Also available are combined Thermo Scientific protease and phosphatase inhibitor cocktails and tablets. These prevent protein degradation and preserve phosphorylation simultaneously, providing complete protection in a single solution or tablet.

All Halt inhibitor cocktails and Pierce inhibitor tablets are compatible with Thermo Scientific Pierce protein extraction reagents and most homemade and commercial cell lysis solutions.

Watch this video to learn more about phosphatase and protease inhibitor tablets

Recommended reading

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