Jun. 09, 2025
In molecular biology Proteinase K (also known as protease K or endopeptidase K) is a broad-spectrum serine protease. The enzyme was discovered in in extracts of the fungus Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest native keratin (hair), hence the name Proteinase “K”. The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to peptidase family S8. The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa).
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WHAT IS THE FUNCTION OF PROTEINASE K IN DNA EXTRACTION?
During the extraction of DNA (or nucleic acids in general), there are many contaminating proteins present. These contaminants must be removed. Proteinase K, which is a broad spectrum serine protease, is used in many DNA extraction protocols to digest these contaminating proteins.
In addition, there may be nucleases (enzymes that degrade nucleic acids) present. The addition of proteinase K degrades these nucleases and protects the nucleic acids from nuclease attack. In addition, proteinase K is stable over a wide pH range and is well suited for use in DNA extraction.
WHAT ARE THE APPLICATIONS OF PROTEINASE K?
Proteinase K is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid. Addition of proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification. It is highly-suited to this application since the enzyme is active in the presence of chemicals that denature proteins, such as SDS and urea, chelating agents such as EDTA, sulfhydryl reagents, as well as trypsin or chymotrypsin inhibitors. Proteinase K is used for the destruction of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases.
Proteinase K is very useful in the isolation of highly native, undamaged DNAs or RNAs, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, particularly in the presence of 0.5 – 1% SDS. Purification of genomic DNA from bacteria (miniprep): bacteria from a saturated liquid culture are lysed and proteins are removed by a digest with 100 μg/ml Proteinase K for 1 h at 37 °C. The enzyme’s activity towards native proteins is stimulated by denaturants such as SDS. In contrast, when measured using peptide substrates, denaturants inhibit the enzyme. The reason for this result is that the denaturing agents unfold the protein substrates and make them more accessible to the protease.
WHY IS PROTEINASE K DIGESTION PERFORMED AT 50°C?
Proteinase K activity is greatly increased by the addition of denaturing agents like SDS or urea (Hilz et al., ), indicating that the denaturation of the substrates helps proteinase K to degrade them. Increasing the temperature to 50°C will also unfold some proteins, making it easier for the proteinase K to degrade them. The proteinase K seems to be a pretty stable enzyme, and can still work at this temperature.
IS PROTEINASE K INACTIVATED BY TEMPERATURE?
Proteinase K is inactivated by heat (e.g. incubating at 55°C).
WHAT IS THE QUICKEST MOST EFFECTIVE WAY TO INACTIVATE PROTEINASE K?
As with most protein enzymes, change the temperature or change the pH significantly.
WHAT IS THE ENZYME ACTIVITY OF PROTEINASE K?
Activated by calcium (1 – 5 mM), the enzyme digests proteins preferentially after hydrophobic amino acids (aliphatic, aromatic and other hydrophobic amino acids). Although calcium ions do not affect the enzyme activity, they do contribute to its stability. Proteins will be completely digested, if the incubation time is long and the protease concentration high enough. Upon removal of the calcium ions, the stability of the enzyme is reduced, but the proteolytic activity remains. Proteinase K has two binding sites for Ca2+, which is located close to the active center, but is not directly involved in the catalytic mechanism. Removal of the Ca2+ ions reduces the catalytic activity of Proteinase K by 80 %. The residual activity is sufficient to digest proteins, which usually contaminate nucleic acid preparations. Therefore, the digest with proteinase K for the purification of nucleic acids is performed in the presence of EDTA (inhibition of magnesium-dependent enzymes). If the presence of Ca2+ required, Ca2+ is added up to a concentration of 1 mM and is removed by the addition of EGTA (pH 8.0; final conc. 2 mM) later on.
WHAT ARE BUFFERS ACCORDING TO PROTEINASE K ACTIVITY?
WHAT ARE THE GUIDELINES FOR USING PROTEINASE K?
1.Isolation of high molecular weight DNA
Chromosomal DNA that has been embedded in agarose plugs can be treated with proteinase K to inactivate rare-cutting restriction enzymes used to digest the DNA. Proteinase K is used for this method at a concentration of 1 mg/ml in a buffer containing 0.5M EDTA and 1% N-lauroylsarcosine (v/v). Incubate 24-48 hours at 37°C.
2.Isolation of plasmid and genomic DNA
Genomic or plasmid DNA can be isolated from liquid nitrogen frozen cells or cultured cells using proteinase K. Incubate 50-100 mg of tissue or 1×108 cells in 1 ml of buffer containing 0.5% SDS (w/v) with proteinase K at a concentration of 1 mg/ml, for 12-18 hours at 50°C.
3.Isolation of RNA
For cytoplasmic RNA isolation, centrifuge the cell lysate, remove the supernate and add 200 ug/ml proteinase K and SDS to 2% (w/v). Incubate for 30 minutes at 37°C. Total RNA can be isolated by passing the lysate through a needle fitted to a syringe prior proteinase K treatment.
4.Inactivation of RNases , DNases and enzymes in reactions
Proteinase K is active in a wide variety of buffers. The enzyme should be used at a ratio of approximately 1:50 (w/w, proteinase K:enzyme ). Incubation is at 37°C for 30 minutes.
HOW DO YOU DETERMINE IF THE PROTEINASE K IS WORKING?
One can use an artificial substrate like benzoyl arginine -p-nitroanilide that when cleaved by the proteinase yields a yellow colored p-nitroaniline that absorbs at ~ 410 nm. You can then determine the activity of the proteinase K by determining how many micromoles of the p-nitroanilide are produced per minute. Then, by dividing by the total amount of protein in the solution, you can determine the specific activity of the enzyme = units (one unit equals 1 mole of p-nitroanilide produced/min ), specific activity = units of enzyme activity/mg total protein. Alternatively, prepare a 1.25 % agar containing 2% casein in pH eight buffer and pour into a petri dish. Punch 4 mm diameter wells in the gel about 20 mm apart. In the wells place various concentrations of your proteinase K solution. Allow to incubate at room temp (humidified) for 6-8 hrs. Look for the clear zones around the wells. The size of the clear zone is proportional to the concentration of the proteinase K and gives a visual appraisal of active digestion of a protein rather than a synthetic substrate.
At Bitesize Bio, we share a lot of troubleshooting tips for RNA and genomic DNA extraction because almost everything we do in molecular biology requires nucleic acid isolation as the very first step. These days, most labs use commercial nucleic acid extraction kits based on spin-column technology.
Genomic DNA extraction kits are generally much easier and faster to use than traditional methods and don’t require significant expertise. The downside, however, is that troubleshooting may be difficult if you don’t understand what is in your kit’s black box!
In this article, we will go through the principles of nucleic acid extraction kits step-by-step. We will also look at some common issues with silica columns that you can overcome with a few simple tips!
Lysis formulas may vary depending on whether you want to extract DNA or RNA. Generally speaking, lysis buffers contain a high concentration of chaotropic salts. Chaotropes have two important roles in nucleic acid extraction:
Chaotropic salts include guanidine HCL, guanidine thiocyanate, urea, and lithium perchlorate.
In addition to chaotropes, a detergent is often present in the lysis buffer to aid protein solubilization and cell lysis.
Enzymes may also feature here, depending on the sample type. The broad-spectrum serine protease Proteinase K is very efficient in digesting proteins away from nucleic acid preparations. Proteinase K works best under protein denaturing conditions (i.e., in denaturing lysis buffer).
Another popular enzyme here, lysozyme, does not work under denaturing conditions and will be most active before the addition of denaturing salts.
Bear in mind that lysis for plasmid isolation is very different from lysis for RNA or genomic DNA extraction because plasmids must be separated from genomic DNA first.
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The addition of chaotropes will release all types of DNA at once, losing the ability to differentiate small circular DNA from high molecular weight chromosomes. Therefore, in plasmid preps, the chaotropes are not added until after cell lysis. For additional reading, check out these great articles on alkaline lysis and plasmid and genomic DNA extraction.
As discussed above, chaotropic salts are critical for lysis and binding to the column. The addition of ethanol (or sometimes isopropanol) will further enhance and influence the binding of nucleic acids to the silica.
Spin columns contain a silica resin that selectively binds DNA (or RNA), depending on salt conditions and other factors influenced by the extraction method. The result is a high-quality material for cloning, long-range sequencing, and long-read sequencing, to name a few potential applications.
Note that the percentage and volume of ethanol used are important. Too much ethanol will bring down degraded material and small species that will influence absorbance at 260 nm (A260 readings). On the other hand, too little ethanol may impede the washing of the salt from the membrane.
Fortunately, the amount of ethanol added will be optimal for the nucleic acid extraction kit you are using. However, if you suspect that degraded DNA is inflating your A260 readings, you can consider re-optimizing the ethanol concentration.
Another useful tip is to save the flow-through and precipitate it to see if you can find your lost material. If you used an SDS-containing detergent for lysis, try using NaCl as a precipitant to avoid detergent contamination of your nucleic acids.
After centrifuging your lysate through the silica membrane, the desired nucleic acids should be bound to the column, and impurities such as protein and polysaccharides should be in the flow-through. However, the membrane will contain protein and salt residues.
At this point, plant samples will likely contain polysaccharides and pigments, while for blood samples, the membrane may be slightly brown or yellow in color. The wash steps remove such impurities.
There are typically two wash steps, although this varies depending on sample type. The first wash will often include a low concentration of chaotropic salts to remove residual proteins and pigments. This is always followed by an ethanol wash to remove the salts.
If the sample didn’t contain a lot of protein starting out (e.g., plasmid preps or PCR clean-ups), an ethanol wash is sufficient.
Removal of the chaotropic salts is crucial to getting high yields and purity. Some kits actually recommend two ethanol washes. Residual salt will impede the elution of nucleic acid, resulting in poor yield, high A230 readings, and thus low A260/230 ratios.
Most protocols include a centrifugation step after washing to dry the column of residual ethanol, and this step is essential for a clean eluent. Subsequent addition of 10 mM Tris buffer or water to the membrane will hydrate the nucleic acids, thus eluting them from the membrane.
Residual ethanol on the membrane at this point will prevent full hydration and elution of nucleic acids.
You will not be able to see ethanol on a spectrophotometer, but a good indicator of its presence is samples that will not sink into the wells of an agarose gel, even in the presence of loading dye. Another indicator of ethanol contamination is samples that don’t freeze at -20°C.
The final step in the DNA extraction protocol is the release of pure DNA or RNA from the silica.
For DNA extraction, 10 mM Tris at pH 8-9 is typically used. DNA is more stable at a slightly basic pH and will dissolve faster in a buffer than in water. This is true even for DNA pellets.
Water tends to have a lower pH of 4-5, and high molecular weight DNA may not completely rehydrate in the short time used for elution. For maximal DNA elution, allow the buffer to stand in the membrane for a few minutes before centrifugation.
For applications requiring intact high molecular weight DNA, such as long-range sequencing and long-read sequencing, elution buffer is the best choice.
RNA, however, can tolerate a slightly acidic pH and dissolves readily in water, making this the preferred diluent.
If you experience lower yields than you expect, there are many factors to consider. It is often a lysis issue, with incomplete lysis being a major cause of low yields. Incorrect binding conditions are another possibility. Make sure to use fresh, high-quality ethanol (100%, 200 proof) to dilute buffers and for the binding step.
Low-quality ethanol or old stocks may have taken up water, skewing the actual working concentration. Remember that if you make your wash buffer incorrectly, you may be washing away your extracted DNA or RNA!
If the extract is contaminated with protein, you may have started with an excess sample, increasing the risk of incomplete solubilization. If the extracts have poor A260/230 ratios, the issue is usually residual salt after binding or inadequate washing. Be sure to use the highest quality ethanol to prepare wash buffers, and if the problem continues, perform an additional wash step.
Environmental samples are especially prone to impurities because humic substances solubilize easily during extraction. Such substances often behave similarly to DNA during the extraction process and are difficult to remove from the silica column. For samples prone to impurities, specialized techniques exist to remove interfering protein and humics prior to column binding.
This is a greater concern for RNA than DNA extraction, and you can find specific advice on troubleshooting RNA extraction here. RNA degradation often occurs due to improper sample storage or inefficient lysis, assuming, of course, samples are eluted with RNase-free water.
For DNA extractions, degradation is not a huge issue if PCR is the desired application, but if you were hoping for intact high molecular weight DNA for long-range sequencing applications you should ensure to not be too harsh when lysing your sample!
PCR cleanup isn’t a DNA extraction technique per se, but it is worth a mention here. Typically, PCR products are cleaned up by adding 3-5 volumes of salt per volume of the PCR reaction, followed by centrifugation of the mixture through a spin column.
Although a failed clean-up is often caused by an unsuccessful PCR, it is worth saving your flow-through after column binding. If a strong PCR band didn’t make it through the column, chances are it is in the flow-through. You can always rescue it and clean it up again.
As scientists, we often want to be able to troubleshoot without asking for outside help. This article should clarify some of the science around silica spin filter technology in many nucleic acid extraction kits allowing you to troubleshoot in no time.
If all else fails, you will have done your homework by the time you call for technical support, and you should reach a resolution much faster, even if that is a free replacement DNA extraction kit!
Do you have any comments or questions about how nucleic acid extraction kits work? Leave a comment below.
Originally published June 28, . Reviewed and republished May and March .
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