Protocol: Protease and Phosphatase Inhibitors

Protease Inhibitors:

InhibitorMWTarget ClassTypeSolubility
Typical Working (1X) Conc.
AEBSF•HCl 239.5 Serine proteases Irreversible 200mg/ml (H2O) 0.2 to 1.0 mM
Aprotinin 6511.5 Serine proteases Reversible 10mg/mL (H2O) 100 to 200nM
Bestatin 308.4 Amino-peptidases Reversible 5mg/mL (MeOH) 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 (H2O) 2 to 10mM
Leupeptin 475.6 Serine and cysteine proteases Reversible 1mg/mL (H2O) 10 to 100µM
Pepstatin A 685.9 Aspartic acid
Reversible 1mg/mL (MeOH) 1 to 20µM
PMSF 174.2 Serine proteases Reversible 18mg/mL (MeOH) 0.1 to 1.0mM

Source: Thermo Scientific

Protease Inhibitor Cocktails:

These are available from many companies. The choice of cocktail depends on the species of sample with which you are working. The cocktails also vary on the presence or absence of EDTA. EDTA is a valuable protease inhibitor, but should be avoided if benzonase is used in sample preparation or if phosphopeptides are to be analyzed. The cocktail may be in a concentrated solution, usually dissolved in DMSO, or a tablet.

Thermo Scientific

Halt Protease Inhibitor Cocktail - AEBSF, aprotinin, bestatin, E-64, leupeptin and pepstatin A, with EDTA (catalog no. 78430) or no EDTA (catalog no. 78425)


Sigma carries a wide variety of cocktails for various species; a useful one is catalog no. P8340

GE Healthcare

Protease inhibitor cocktail, catalog no. GE80-6501-23, has no EDTA


Complete protease Inhibitor cocktail tablets, EDTA free, 1 tablet for 10 ml, catalog no. 11836170001


Protease Inhibitor Cocktail, a powder that can be dissolved in DMSO to make a 500 x solution. Contains Aprotinin, Leupeptin, Pepstatin, PMSF. Catalog no. ab65621

Phosphatase Inhibitors:

Alkaline phosphatase Imidazole, sodium orthovanadate, (-) p-bromotetramisole oxalate, levamisole
Acid Phosphatase NaF, sodium molybdate, sodium tartrate
Ser/Thr Phosphatases NaF, beta-glycerophosphate, sodium pyrophosphate, cantharidin, okadaic acid, EDTA, cyclosporin, FK-506, calyculin A
Tyrosine phosphatases Sodium orthovanadate, peroxovanadium compounds

Phosphatase Inhibitor Cocktails:


  • Phosphatase Inhibitor Cocktail 2 - sodium vanadate, sodium molybdate, sodium tartrate, and imidazole (catalog no. P5726)
  • Phosphatase Inhibitor Cocktail 3- cantharidin, p-bromolevamisole oxalate, and calyculin A (catalog no. P0044)

Sigma recommends using Cocktail 2 and Cocktail 3 in combination

EMD Millipore

  • Phosphatase Inhibitor Cocktail Set I (catalog no. 524624) - (-)-p-bromotetramisole, cantharidin, microcystin LR
  • Phosphatase Inhibitor Cocktail Set II (catalog no. 524625) - imidazole, NaF, sodium molybdate, sodium orthovanadate, sodium tartrate

Thermo Scientific

  • Halt Phosphatase Inhibitor Cocktail - sodium fluoride, sodium orthovanadate*, sodium pyrophosphate and beta-glycerophosphate. Thermo Scientific 78428
  • Halt Protease and Phosphatase Inhibitor Cocktail - sodium fluoride, sodium orthovanadate*, sodium pyrophosphate and beta-glycerophosphate, aprotinin, bestatin, E-64, leupeptin (Thermo catalog no. 78442, with EDTA or catalog no. 78443, without EDTA)

*Sodium orthovanadate preparation

All steps to be performed in a fume hood.

  • Prepare a 100 mM solution in double distilled water.
  • Set pH to 9.0 with HCl.
  • Boil until colorless. Minimize volume change due to evaporation by covering loosely.
  • Cool to room temperature.
  • Set pH to 9.0 again.
  • Boil again until colorless.
  • Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling.
  • Bring up to the initial volume with water.
  • Store in aliquots at - 20°C. Discard if samples turn yellow

Source: Abcam