With so many brands of protease and phosphatase inhibitors, it can be difficult to know which ones best suit your experimental needs. This article will provide tips about how to choose inhibitors and how to use them in the lab.
Proteases and phosphatases are essential enzymes for a variety of biochemical pathways. Proteases break proteins into smaller polypeptides or amino acids during the process of proteolysis. Meanwhile, phosphatases remove phosphate groups from proteins―performing the opposite action of kinases.
In an intact cell, protease and phosphatase enzymes are typically sequestered in cellular compartments. However, during the process of cell lysis, these compartments are broken open, releasing proteases and phosphatases that may degrade proteins and cause changes in phosphorylation states. To prevent these alterations, various protease and phosphatase inhibitors are often added to lysis buffers prior to cell membrane rupture.
Commercial cocktails are available that inhibit a wide variety of both protease and phosphatase enzymes during cell lysis and protein purification. Alternatively, other cocktails are available that inhibit only proteases or phosphatases.
In general, protease inhibitors are nearly always needed during cell lysis and protein purification procedures.1 In contrast, phosphatase inhibitors are only required when phosphorylation states are analyzed, usually to study activation states.1
Ultimately, the purpose of a phosphatase inhibitor is to protect proteins from dephosphorylation during lysis and extraction procedures. This allows the phosphorylation state to be preserved. Phosphatase inhibitors may be necessary to analyze signaling cascades, protein-protein interactions, or phosphorylated sites on proteins.1 Otherwise, you probably do not need a phosphatase inhibitor.
Researchers can begin by purchasing either individual protease inhibitors (such as AEBSF, aprotinin, bestatin, E-64, EDTA, and leupeptin) or cocktail combinations that contain multiple inhibitors. For most cell lysis and purification procedures, it is recommended to start with a protease inhibitor cocktail.2 Then, if there is evidence of downstream issues (such as the lack of full-length proteins), specific protease inhibitors can be considered.2
There are many brands of protease cocktail inhibitors. Some are optimized for bacterial, mammalian, plant, and yeast research, while others are specific for purifying histidine-tagged proteins.3 Other protease inhibitor cocktails are designed to be fully compatible with mass spectrometry.4 However, it is essential to note that there is no universal inhibitor that is guaranteed to inactivate all proteases.2
A final key question involves whether your protease inhibitor cocktail should contain EDTA. The presence of EDTA is problematic if you intend to purify your protein of interest with immobilized metal affinity chromatography (IMAC). Furthermore, EDTA may not work well if your extraction buffers contain divalent cations like Ca2+, Mg2+, or Mn2+.5 Finally, EDTA is generally not compatible with two-dimensional gel electrophoresis.2
Protease and phosphatase inhibitors should be added to your lysis buffer immediately before cell lysis. Because these inhibitors are generally not stable for long periods of time, all working solutions should be prepared immediately before use. Furthermore, it is imperative that all inhibitors are stored according to the manufacturer’s instructions.
It is recommended that protease inhibitors be added to all buffers in which proteases may be present.2 Some protease inhibitors, such as PMSF, are particularly unstable and may need to be added multiple times during lysis and purification to ensure protease inhibition.2
Another consideration is whether to purchase inhibitors in a tablet, powder, or liquid form. Many researchers prefer tablets because they can then be conveniently dropped into a specific volume of buffer without pipetting.2
In addition to the judicious use of protease inhibitors, it is also essential that cell lysis be carried out in an optimal way.2 Cell lysis and purification should generally be performed on ice or at 4°C, which slows down the activity of any remaining proteases.2
Most cell lysis experiments are also performed at a neutral or slightly alkaline pH that will minimize the activity of acid proteases.6 Furthermore, most inhibitor cocktails are prepared in stock solutions at a pH of around 7.5
Some companies sell protease activity assays that allow researchers to check whether protease inhibition is occurring as desired.2 These assays allow for the rapid and highly sensitive analysis of trace protease activity using a spectrophotometer. One such assay monitors the degradation of casein, the protein that is responsible for the white color of milk.7
Researchers can use protease activity assays on samples with and without a protease inhibitor to assess how well their protease inhibitors work. However, before resorting to these assays, it may be easier to prepare fresh protease inhibitor solutions if protease inhibition issues are suspected.2
MilliporeSigma provides various protease and phosphatase inhibitor products, including different cocktail combinations. For more information, visit this page.
PhosSTOP™
cOmplete™, EDTA-free Protease Inhibitor Cocktail
1. Gleiser I, Yaish P, Barnea-Gedalyahu E, Zharhary D. Phosphatase Inhibitor Cocktail. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/protein-lysis-and-extraction/phosphatase-inhibitor.
2. Ritchie C. Protease inhibitors. February 12, 2013. www.labome.com. https://www.labome.com/method/Protease-Inhibitors.html.
3. Protease Inhibitor Cocktails. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/protein-biology/protein-lysis-and-extraction/protease-inhibitor-cocktails.
4. MS-SAFE Protease and Phosphatase Inhibitor. Mass Spectrometry Safe Protease and Phosphatase Inhibitor Cocktail. Product information. www.sigmaaldrich.com. Reviewed March 2022. https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/137/142/mssafebul-ms.pdf.
5. cOmplete™ Protease Inhibitor Cocktail. www.sigmaaldrich.com. https://www.sigmaaldrich.com/US/en/product/roche/coro.
6. Ryan BJ, Henehan GT. Avoiding proteolysis during protein purification. Methods Mol Biol. 2017;1485, 53–69.
7. Universal Protease Substrate. www.sigmaaldrich.com. https://www.sigmaaldrich.com/US/en/product/roche/11734334001.