A lot of questions pop up when it comes to using proteinase K, such as how to inactivate proteinase K, how should proteinase K be stored, what is the solubility of proteinase K… The list goes on. We decided to compile the most important questions and turn this page into the go-to resource for the product. Here is some important information about proteinase K.

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1.How do you inactivate proteinase K?

Inactivating proteinase K is perhaps one of the most common questions we see. And the answer is very simple. Heat is a widely used way of inactivating proteinase K. While the activity of proteinase K increases with temperature, and is optimized at about 65 ˚C, heating proteinase K to 95 ˚C for 10 minutes will inactivate it. Keep in mind, however, that heating proteinase K does not fully inactivate the enzyme. There will always be a small amount of activity remaining through this method.

Protease inhibitors such as PMSF and AEBSF (Pefabloc®) can also be used to permanently inactivate proteinase K.

Note- the actual inactivation temperature has been debated, ranging between 70 - 95 ˚C. However, crowd sourced feedback and extensive research led us to settle on 95 ˚C as the best temperature for inactivation.

2.What is the optimal temperature for proteinase K activation?

As stated above in #1, proteinase K activity increases with temperature (up to a certain point). The optimal temperature for activity ranges between 50-65 ˚C. The higher temperatures help with protein unfolding, easing the ability for proteinase K to breakdown those proteins.

But optimizing your proteinase K might not be the most important thing during your procedure. Sometimes, special techniques will require adapted temperatures to yield the best overall results. Therefore, something else to keep in mind is that while the listed range is great for proteinase K activity, the enzyme is still active in temperatures ranging between ~20-65 ˚C, and having that wide temperature flexibility available might be useful for very particular methods you’re performing. Beyond 65 ˚C, as temperatures increase, you risk inactivating proteinase K.

3.What exactly is the relationship between proteinase K and calcium?

Proteinase K binds to two Ca 2+ ions which help maintain the stability of the enzyme, especially when it’s subjected to increasing temperatures. Calcium also protects proteinase K from autolysis. While calcium helps maintain proteinase K thermostability, it is not necessary for proteolytic activity.

According to Richard Tullis and Harvey Rubin, this relationship becomes more interesting when DNase I is involved. Proteinase K is known to inactivate DNases and RNases, but when DNase I is in the presence of Ca2+, it is protected from proteinase K (concentration of 1mg/ml). RNase, on the other hand, is inactivated whether or not it is in the presence of Ca2+. Their findings suggest a method for treating contaminated RNase free DNaseI or isolating highly polymerized RNA.

4.Does EDTA inactivate proteinase K?

This question also seems to come up quite a bit when discussing proteinase K. Chelators such as EDTA or EGTA don’t have a direct effect on proteinase K enzyme activity.

Often, the reason for using EDTA with proteinase K during DNA or RNA purification is for the removal of calcium (see #3). But because calcium is related to proteinase K stability, the addition of EDTA can impact the calcium and therefore reduce proteinase K activity to some extent.

Proteinase K Free Handbook - get protocols, troubleshooting, helpful articles and more inside.

5.What are the activators of proteinase K?

Proteinase K activators include SDS (sodium dodecyl sulfate) and urea. Generally, proteinase K becomes more stable and more active when in buffers that contain these activators.

6.How is proteinase K involved with cell lysis?

To answer this question, the first thing to consider is what proteinase K is. It’s a broad-spectrum protease capable of digesting a wide range of native proteins (more details are later in this article).

When it comes to cell lysis, particularly for downstream DNA isolation and purification, proteinase K can be part of the lysis step by digesting surface proteins. Further into the procedure, when it comes time to resuspend and lyse the nuclei in a buffer containing proteinase K, the proteinase K will help digest proteins that would otherwise degrade the sample.

7.Why do many DNA extraction lysis buffer recipes call for proteinase K and RNase?

This question comes up every now and then because the suggestion seems like a contradiction. Proteinase K is known to digest RNases, so why would the two be added together in a lysis buffer? First of all, you want the RNase added because it would break down contaminating RNA during your DNA isolation. And you want to use proteinase K because it will break down damaging proteins, DNases and RNases.

The answer to this question is really rooted in timing and optimization. Some researchers suggest adding the RNase in first, allowing time for it to work. Then proteinase K and SDS can be added to break down unwanted proteins. Something else to consider is that some protocols will have you incubate in SDS, proteinase K and RNase at 37 ˚C for a certain amount of time. Since proteinase K activity is not as highly optimized at this temperature, this likely gives RNase time to work. Later steps in the protocol suggest a second incubation at 55 ˚C for a longer period of time, which would be a more optimal temperature for proteinase K activity, allowing it to digest other unwanted proteins.

8.Why is knowing the activity of proteinase K important?

We actually dedicated a whole article to explaining more about proteinase K activity and concentration. The scenario we presented was a situation where a researcher uses an old protocol that calls for 1µl of proteinase K. While the protocol calls for that volume, she might actually require more or less of her enzyme, depending on enzyme activity. In the end, the article highlights important information defining enzyme activity, specific activity and other ways to evaluate proteinase K.

9.What applications is proteinase K used for?

Proteinase K is a broad-spectrum serine protease under the subtilisin-like class (there are two types of serine proteases, chymotrypsin-like and subtilisin-like). As a broad-spectrum protease, it is primarily used for the isolation of nucleic acids: genomic DNA, cytoplasmic RNA, highly native DNA and RNA, etc. It is ideal for these applications because proteinase K is able to break down proteins and inactivate DNases and RNases that would otherwise degrade a desired sample of DNA or RNA. To list it out, making this simpler to follow, proteinase K is used for:

  • Digestion of unwanted proteins in molecular biology applications
  • Removal of endotoxins bound to cationic proteins such as lysozyme and RNaseA
  • Removal of nucleases for in situ hybridization
  • Prion research with respect to TSE (transmissible spongiform encephalopathies)
  • Protease footprinting
  • Mitochontrial isolation
  • Isolation of genomic DNA
  • Isolation of cytoplasmic RNA
  • Isolation of highly native DNA or RNA

Proteinase K Handbook

10.How should Proteinase K be stored/ What is the shelf-life of Proteinase K?

Stock Solution: aliquot your stock solution and store at -20 ˚C for up to 1 year.

Lyophilized Powder: Store desiccated at -20 ˚C for up to 2 years.

11.How do you make proteinase K stock solution?

Use our protocol for making a 20 mg/ml stock solution of proteinase K using Tris buffer and CaCl2.

12.How much solution would 1 gram of your proteinase K make?

In a 20 mg/ml solution, the solution equivalents would be as follows:

Pack Sizes Solution Equivalent
100 mg 5 ml (20 mg/ml)
500 mg 25 ml (20 mg/ml
1 g 50 ml (20 mg/ml

This is with an activity of 30 units/mg and 600 AU/ml in a 20 mg/ml solution.

13.What can proteinase K be dissolved in / How do you dissolve proteinase K?

Proteinase K is very water soluble, and it can also be dissolved in Tris or PBS.

When working with PBS, however, it can be a little tricky, which is possibly due to the pH (still within optimal range, but on the lower end of that range). Typically, adding proteinase K powder a little at a time while mixing into solution will help dissolve it into PBS.

14.How do I inactivate nucleases with proteinase K?

What proteinase K is known for is protecting nucleic acids from protein degradation. This occurs because proteinase K is capable of digesting proteins that would normally damage your sample. This protocol further details how to use proteinase K to inactivate nucleases during your extraction procedures.

15.Is there an alternative to using proteinase K during DNA extractions?

The benefit of using proteinase K during DNA extraction is its ability to degrade a wide range of damaging nucleases. It’s also great for digesting surface proteins on the cell membrane. However, if this question is specifically geared toward isolating DNA from other proteins, the phenol-chloroform extraction is another option useful for removing proteins from solution. However, this method is more toxic.

16.What does proteinase K have to do with prion diseases or TSE?

Proteinase K is involved in differentiating between the normal PrP C (prion protein / protease-resistant protein) and PrPSC (disease causing isoform). Both PrPC and PrPSC have the same molecular weight, however, PrPSC is resistant to proteinase K. Samples, which might contain both, are treated with proteinase K, which will eliminate PrPC and convert PrPSC into PrPRES which has a lower molecular weight and can be pelleted, and therefore distinguished.

17.What is the molecular weight of proteinase K?

The molecular weight of proteinase k is 28.5 kDa.

18.What is the optimal pH for proteinase K?

Proteinase K is active in a pH between 7.5 and 12.0.

19.What is the primary sequence for proteinase K?







20.What is Proteinase K?

We decided to save this question for last, mainly because that answer can be found easily on our website, and it’s hinted throughout this page. However the question absolutely deserves a defined space here.

Proteinase K is a broad-spectrum serine protease within the subtilisin family of proteins. It’s well-known within research for its ability to inactivate RNases and DNases that would damage desired nucleic acid samples during extraction. It got its name because of its originally discovered ability to hydrolize keratine.

    Proteinase K Handbook


    Breyer, J., Wemheuer, W. M., Wrede, A., Graham, C., Benestad, S. L., Brenig, B., . . . Schulz-Schaeffer, W. J. (2012). Detergents modify proteinase K resistance of PrPSc in different transmissible spongiform encephalopathies (TSEs). Veterinary Microbiology, 157(1-2), 23-31. doi:10.1016/j.vetmic.2011.12.008

    Charette, S. J., & Cosson, P. (2004, September). Quick preparation of genomic DNA for PCR analysis. Retrieved May 06, 2016, from http://www.dictybase.org/techniques/molec_biol/qui...

    Ebeling, W., Hennrich, N., Klockow, M., Metz, H., Orth, H., & Lang, H. (1974). Proteinase K from Tritirachium album Limber. Eur. J. Biochem, 47, 91-97. Retrieved May 05, 2016, from http://onlinelibrary.wiley.com/doi/10.1111/j.1432-...

    Muller, A., Hinrichs, W., Wolf, W. M., & Saenger, W. (n.d.). Crystal structure of calcium-free proteinase K at 1.5-A resolution. The Journal of Biological Chemistry, 269, 23108-23111. Retrieved May 04, 2016, from http://www.jbc.org/content/269/37/23108.short

    Tullis, R. H., & Rubin, H. (1980). Calcium protects DNase I from proteinase K: A new method for the removal of contaminating RNase from DNase I. Analytical Biochemistry, 107(1), 260-264. doi:10.1016/0003-2697(80)90519-9

Karen Martin
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"To understand the universe is to understand math." My 8th grade
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