How to Select the Right Protease Inhibitor

Finding the right combination of protease inhibitors for a given experiment can be complicated. Furthermore, how do you know whether your protease inhibitors are working as intended? Biocompare recently asked experts from two leading companies—MilliporeSigma and Thermo Fisher Scientific—for some tips.

What are protease inhibitors?

According to Halina Zakowicz, Global Market Development Specialist in Protein Preparation at Thermo Fisher Scientific, all living organisms contain a variety of enzymes called proteases that break down proteins into peptides and amino acids—in a process called proteolysis.

In whole cells, proteases are confined to compartments. However, these compartments rupture during the process of cell lysis, releasing proteases that may degrade proteins. “Following cell lysis, you often end up with a reduced recovery of total proteins and a biologically meaningless representation of protein activities,” Zakowicz explains.

Zakowicz adds that protease inhibitors, which are typically added to lysis buffers, bind to proteases and prevent proteins from being degraded during cell lysis. She notes that protease inhibitors are often added to cell lysates for applications such as western blotting, immunoprecipitation, mass spectrometry, 2D gel electrophoresis, and protein-protein interaction studies.

Tips for choosing protease inhibitors

While it is possible to buy individual protease inhibitors, Eleni Sommerkamp, Product Manager of Protein Separation at MilliporeSigma, recommends cocktails that combine multiple inhibitors in one product. “Unless a specific protease is being targeted, it is usually best to use a broad-spectrum cocktail for the preservation of the target,” she advises.

Zakowicz says that most protease cocktails—including Thermo Scientific’s Halt™ Protease Inhibitor Cocktail (100X)—contain six inhibitors (AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstatin A) with and without EDTA. Some researchers also choose to add PMSF. “However, it is important to understand that there is no single protease inhibitor cocktail that will stop all proteases from degrading proteins,” she warns.

MilliporeSigma sells a wide variety of different protease inhibitors that can be added to solutions before cell lysis and protein purification steps. “Some of our cocktails are specific for bacteria, yeast, or mammals, while other products can be added directly to tissue and cell culture media,” Sommerkamp notes.

Another consideration is whether to purchase inhibitors in a tablet, powder, or liquid form. Sommerkamp recommends MilliporeSigma’s new ReadyShield^® Inhibitor Cocktail product line. “The great thing about these cocktails is that they do not freeze in the freezer or on ice. They are always ready to use, and you don’t have to dethaw them.”

Do you need EDTA, DMSO, and phosphatase inhibitors?

Zakowicz notes that protease inhibitor cocktails are sold with or without EDTA and DMSO. She warns that EDTA—used as a metalloprotease inhibitor—is incompatible with nickel-based chromatography columns due to its affinity for divalent cations. She also notes that the presence of EDTA in a protease inhibitor cocktail may inhibit other cellular proteins. “Testing may be necessary to determine if an EDTA-free formulation of the protease inhibitor is needed.”

Zakowicz further explains that DMSO is a stabilizer and solvent frequently used in storing and packing biological compounds. “At low concentrations, DMSO is not expected to adversely affect molecular activities," she says. “However, at higher concentrations, DMSO may precipitate or denature proteins and enzymes or reduce their catalytic activities.”

If there is a concern about the presence of EDTA or DMSO in the biological sample, both compounds can be removed by a dialysis or desalting protocol. However, it is generally easier to avoid adding them in the first place if you think they might be incompatible with your work. Zakowicz recommends using Thermo Scientific’s Pierce™ Protease Inhibitor Tablets because they are available in EDTA- and DMSO-free formats.

Zakowicz explains that phosphatase inhibitors are sometimes added along with protease inhibitors. Phosphatase inhibitors prevent the degradation by phosphatases during cell lysis or extraction. “However, phosphatase inhibitors are typically only used when phosphorylation states―or in other words, activation states―are being investigated.”

Like protease inhibitors, phosphatase inhibitors are provided as cocktail tablets that target a broad spectrum of phosphatases. Combination protease and phosphatase inhibitor cocktails—such as Thermo Scientific’s Halt™ Protease and Phosphatase Inhibitor Cocktail (100X)—are a good choice for dual inhibition, says Zakowicz.

Tips to assess whether your protease inhibitor is working

Zakowicz and Sommerkamp agree about the importance of always reviewing product use guidelines. It is also imperative to follow storage instructions carefully. Zakowicz notes that some inhibitors do not function well at very high or low pH levels.

But how can you assess whether your protease inhibitor is working as desired? Sommerkamp recommends MilliporeSigma’s Universal Protease Substrate. She explains that the assay uses casein labeled with resorufin as the substrate. “If there is protease activity in a sample, casein will be degraded, releasing resorufin. The amount of resorufin released can then be detected spectrophotometrically and fluorometrically.”

Sommerkamp suggests that researchers add the Universal Protease Substrate to samples with and without their protease inhibitor. “The inhibitor should stop proteases from degrading casein and releasing resorufin. This should result in a lower absorbance compared with samples without an inhibitor.”

Finally, Sommerkamp suggests several other methods for analyzing the amount of protein in samples with and without protease inhibitors. Common qualitative methods include western blotting or gel electrophoresis, while quantitative techniques include mass spectrometry and absorbance-related detection methods—like Biuret and Bradford assays. “In all of these cases, a sample that included a protease inhibitor in the upstream steps should have more protein detected or quantified than a sample where no protease inhibitor was used.”