Protease inhibitors and protease inhibitor cocktails are a useful reagent during protein research applications. In this article, we'll take a look at what protease inhibitors and cocktails are, how to prepare your cocktails, when to use them, and so much more. Along with this article is a helpful animation and illustrations.
In This Article:
What is a protease inhibitor cocktail?
In molecular research, protease inhibitor cocktails are a blend of chemicals called proteases inhibitors that protect proteins from degradation by endogenous proteases.
What are proteases (proteolytic enzymes)?
Proteases are enzymes that degrade proteins. Proteases play important roles in protein catabolism. Through their degradative nature, proteases indirectly influence protein activity, protein-protein interaction and the production of bioactive molecules.
Furthermore, proteases are vital for food digestion (digestion of proteins), cell division, blood-clotting cascade, apoptosis and retroviral life-cycle.
Proteases work by hydrolyzing peptide bonds. Peptide bonds are what link amino acids together into proteins. A peptide bond occurs when an acid group of one amino acid comes in close contact with an amino group of the second amino acid. During the reaction, water is eliminated. The carbon from the acid group bonds to the nitrogen of the amino group.
The hydrolysis (in this case, the breaking) of the peptide bond, therefore, occurs through the addition of water. As enzymes, proteases catalyze these hydrolysis reactions between interior peptide bonds.
What are protease inhibitors?
During cell lysis, proteases encounter proteins that they otherwise wouldn’t. For protein purification, the introduction of proteases into an unregulated environment is hazardous to the target protein.
Protease inhibitors protect target proteins from proteolysis. Different protease inhibitors target different types of proteases. Therefore, it’s not uncommon for researchers to use a combination of protease inhibitors, or to use a cocktail of protease inhibitors to optimize protein purification.
How protease inhibitors and cocktails are categorized
Protease are grouped based on the type of proteases they act upon as well as their mechanism on the active site of proteases.
Reversible Protease Inhibitors
Protease inhibitors categorized as reversible can bind to proteases without changing their active site. They can act competitively, that is, they bind to a protease’s active sites and compete with other substrates for the active site.
There are also uncompetitive reversible protease inhibitors. Rather than competing for an active site, these inhibitors bind with the protease only when a substrate is bound to the protease.
Finally, noncompetitive reversible protease inhibitors bind based on affinity to the protease.
Irreversible Protease Inhibitors
Irreversible protease inhibitors change protease active sites. Another term used for irreversible protease inhibitors is inactivators because they inactivate a protease’s active site. The affinity in this mechanism is so strong, that these protease inhibitors can’t be removed, making them irreversible.
One of the pros of using reversible protease inhibitors is that they can be removed. The downside, however, is that their concentration must be maintained throughout the entire process.
Irreversible protease inhibitors offer the benefit of inactivating proteases fully, meaning that their concentration does not have to be continually maintained. However, these inhibitors are not easily removed
How do I prepare my protease inhibitor cocktail?
Prepare your protease inhibitor cocktails according to your supplier’s protocols. Provided here are cocktail preparation examples for some of GoldBio’s protease inhibitor cocktails. You can find all of our protease inhibitor cocktail protocols online by searching GoldBio’s website.
Allow solution to warm to room temperature. The solution is in suspension form, vortex the vial before removing the solution.
Add ProBlock™ Gold 10 μl/ml directly in an appropriate volume of extraction buffer or protein extract to 1X final concentration. For more potent protease inhibition,
ProBlock™ Gold 20-30 μl/ml to give 2-3X final concentration.
* When ProBlock™ Gold is added to the buffer or extract, it is stable for up to week at 4°C and 4-6 weeks at -20°C.
Note: (OPTIONAL). For inhibition of metalloproteases (if the buffer does not contain EDTA), add 0.5M EDTA 10 μl/ml directly in an appropriate volume of extraction buffer or extract to 1X final concentration.
Allow the ProBlock™ Gold Mammalian protease inhibitor cocktail solution to warm to room temperature. This solution is in suspension form. Vortex the vial before removing any solution.
Add ProBlock™ Gold Mammalian protease inhibitor cocktail 10 μl/ml directly to an
appropriate volume of extraction buffer or protein extract to 1X final concentration.
For more potent protease inhibition, add ProBlock™ Gold Mammalian protease
inhibitor cocktail 20-30 μl/ml to give 2-3X final concentration
* When ProBlock™ Gold Mammalian is added to the buffer or extract, it is stable for 1-2 weeks at 4°C and 4-6 weeks at -20°C
Note: (OPTIONAL). For inhibition of metalloproteases (if the buffer does not contain EDTA), add 0.5M EDTA (10 μl/ml) and 1M 1,10-Phenanthroline (10 μl/ml) directly to an appropriate volume of extraction buffer or extract to 1X final concentration (See Table).
Allow the ProBlock™ Gold Extra Strength solution to warm to room temperature. This solution is in suspension form. Vortex the vial before removing any solution.
Add ProBlock™ Gold Extra Strength solution 10 μl/ml directly to an appropriate volume of extraction buffer or protein extract to 1X final concentration. For more potent protease inhibition, add ProBlock™ Gold Extra Strength solution 20-30 μl/ml to give 2- 3X final concentration
* When ProBlock™ Gold Extra Strength is added to the buffer or extract, it is stable for up to week at 4°C and 4-6 weeks at -20°C.
Mix solution thoroughly.
Note: (OPTIONAL). For inhibition of metalloproteases (if the buffer does not contain EDTA),
add 0.5M EDTA (15 μl/ml) and 1M 1,10-Phenanthroline (15 μl/ml) directly to an appropriate
volume of extraction buffer or extract to 1.5X final concentration (See Table).
What volume or concentration of protease inhibitor should I use?
Refer to your supplier’s protocols to determine the best amounts and concentrations of cocktail to use in your lysis buffer.
One important thing to keep in mind is the concentration will also depend on what you’re trying to accomplish with your protease inhibitors and cocktails. For instance, if you’re using this in cell growth media, you’ll want a significantly lower concentration than you would if you are using this in lysis buffer.
Why are premade protease inhibitor cocktails important?
Proteases are enzymes that degrade proteins by hydrolyzing peptide bonds. During cell lysis, these naturally occurring enzymes mix with your desired proteins, which is not ideal for protein purification.
Protease inhibitors prevent proteases from hydrolyzing those peptide bonds. You can find individually sold protease inhibitors such as AEBSF, aprotinin and PMSF. Or you can find protease inhibitor cocktails.
Protease inhibitor cocktails are especially helpful for a number of reasons:
Protease inhibitors are grouped by the proteases they act against. Examples of protease inhibitor classes are serine protease inhibitors (which inhibit serine proteases), aspartic protease inhibits (inhibits aspartic proteases), and cysteine protease inhibitors (inhibits cysteine proteases).There are some cases when using one single protease inhibitor is not enough. In this case, an optimized mixture or “cocktail” of protease inhibitors tailored to your experimental conditions will better protect your proteins.
Mixing your own blend of cocktails can sometimes require further experimentation to perfectly optimize the cocktail to your required conditions. Furthermore, small batch production can sometimes cause minor inconsistencies.
Premade cocktails cut down the time, uncertainty and inconsistency because they have already been developed, tested and optimized. Their production is scaled, which also ensures more batch-to-batch consistency.
Shopping à la carte gets expensive really quick. Let’s say you have identified three protease inhibitors to make your own cocktails. However, you think to yourself, “I’m not even going to use up the whole bottle of any of those. And I’ll be spending $150+ on stuff I’ll be throwing out.”
This is where commercially available protease inhibitor cocktails come in handy. Here you can order an appropriate pack size for your application. You get exactly the amounts of inhibitors you need and you spend far less money and time.
What applications are protease inhibitors and protease inhibitor cocktails used in?
Scientists use protease inhibitors and cocktails in a wide range of research applications and even in drug development. Outside of molecular biology and clinical research, protease inhibitors are vital for natural regulation of living organisms. Here are some examples where protease inhibitors and cocktails are used.
- Protein purification
- Cell function
- Drug development
- Cell culture
- Natural cell regulation
- Microbial defense
At what step do I use protease inhibitors or protease inhibitor cocktails?
When doing protein research, protease inhibitors and cocktails are often used to protect target proteins from protease degradation that would otherwise occur during cell lysis.
Inhibitors and cocktails are generally added to lysate before homogenization (rupturing the cell membrane). If not, proteases will rapidly degrade susceptible proteins.
Some researchers recommend using protease inhibitors and cocktails before you rupture the plasma membrane and after.
Working on ice is another way to limit the potential for degradation. For very sensitive proteins, adding protease inhibitors to thawed cells can be very helpful.
Ultimately, when considering when to add your protease inhibitors or inhibitor cocktail, your goal is to protect your protein at each stage.
- Add protease inhibitors or protease inhibitor cocktails just before homogenization.
- Work on ice.
- Consider adding protease inhibitors before and after plasma membrane rupture.
- Protect your cell and consider every stage where your proteins are vulnerable.
What’s in the protease inhibitor cocktails?
What’s in a protease inhibitor cocktail will depend on one cocktail from another. Commercially available protease inhibitor cocktails will usually list the types of protease inhibitors used.
For instance, GoldBio’s ProBlockTM Gold Protease Inhibitor Cocktail [100x] (GB-108) has been made with a variety of inhibitors, each at optimized concentrations. The formulation includes AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin and PMSF. It also comes with a separately packaged container of EDTA for metalloprotease inhibition.
When shopping for cocktails or making your own, give consideration to what your target protein will require and the different features of each protease inhibitor.
GoldBio Protease Inhibitor Cocktails
- ProBlock™ Gold Protease Inhibitor Cocktail [100X]
- ProBlock™ Gold Bacterial 2D Protease Inhibitor Cocktail [100X]
- ProBlock™ Gold 2D Protease Inhibitor Cocktail (EDTA Free) [100X]
- ProBlock™ Protease Inhibitor Cocktail -100, Plus EDTA
- ProBlock™ Protease Inhibitor Cocktail -100, EDTA free
- ProBlock™ Protease Inhibitor Cocktail -50, Plus EDTA
- ProBlock™ Protease Inhibitor Cocktail -50, EDTA free
- How to Prepare Protein Samples for Western Blot
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- Why is my lysis buffer not working? – How to Resolve 9 Lysis Buffer Issues
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