10 Questions you want to ask about PROTEINASE K

10 Questions you want to ask about PROTEINASE K

  1. QUESTION: What is Proteinase K?


Proteinase K 1

  1. QUESTION: What is the function of proteinase K in DNA extraction?



  1. QUESTION: What are proteinase K applications?


  1. QUESTION: Why is proteinase K digestion performed at 50°C?


  1. QUESTION: What are temperatures proteinase k inactivated?


  1. QUESTION: What is the quickest most effective way to inactivate proteinase K?

ANSWER: As with most protein enzymes change the temperature or change the pH significantly.

  1. QUESTION: What is the Enzyme activity of proteinase K?

ANSWER: 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.


  1. QUESTION: What are buffers according to Proteinase K activity?


Buffer (pH 8.0, 50°C, 1.25 µg/ml protease K, 15 min incubation) Proteinase K activity (%)
30 mM Tris·Cl 100
30 mM Tris·Cl; 30 mM EDTA; 5% Tween 20; 0.5% Triton X-100; 800 mM GuHCl 313
36 mM Tris·Cl; 36 mM EDTA; 5% Tween 20; 0.36% Triton X-100; 735 mM GuHCl 301
10 mM Tris·Cl; 25 mM EDTA; 100 mM NaCl; 0.5% SDS 128
10 mM Tris·Cl; 100 mM EDTA; 20 mM NaCl; 1% Sarkosyl 74
10 mM Tris·Cl; 50 mM KCl; 1.5 mM MgCl2; 0.45% Tween 20; 0.5% Triton X-100 106
10 mM Tris·Cl; 100 mM EDTA; 0.5% SDS 120
30 mM Tris·Cl; 10 mM EDTA; 1% SDS 203
  1. QUESTION: 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 1mg/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 ProteinaseK at a concentration of 1mg/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 200ug/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 (see FAQ “What is the Proteinase K activity in commonly used 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.

  1. QUESTION: How to determine if the proteinase K is working?

ANSWER: One can use an artificial substrate like benzoyl arginine -p-nitroanilide that when cleaved by the proteinase yield sa yellow colored p-nitroaniline that absorbs at ~ 410 nm. You can then determine the activity of the proteinase K by determinh how many micromoles of the p-nitroanilide are produced per minute. The by dividing by the total amount of protein in the solution you can determine the specific activity of the enzyme activity = units ( one unit equals 1 umole 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 4mm diameter wells in the gel about 20 mm apart. In the wells place various concetrations 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 oproortional to the concentration of the proteinase K and gives a visual appraisal of active digestion of a protein rather than a synthetic substrate.

Proteinase K Protein Structure


  1. Betzel C, Singh TP, Visanji M, et al. (July 1993). “Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution”. The Journal of Biological Chemistry 268 (21): 15854–15858. PMID 8340410.
  2. Ebeling W, Hennrich N, et al. (1974). “Proteinase K from Tritirachium album Limber”. European Journal of Biochemistry 47 (1): 91–97. doi:10.1111/j.1432-1033.1974.tb03671.x. PMID 4373242.
  3. Müller A, Hinrichs W, Wolf WM, Saenger W (1994). “Crystal structure of calcium-free proteinase K at 1.5-Å resolution”. The Journal of Biological Chemistry 269: 23108-23111. PMID 8083213.
  4. http://wiki.answers.com/Q/What_is_the_function_of_Proteinase_K_in_DNA_extraction
  5. Wikipedia.

96 thoughts on “10 Questions you want to ask about PROTEINASE K”


  2. To Maria Romero.




    The typical QC method to determine units for Proteinase K is that one unit will hydrolyze urea-denatured hemoglobin to produce color equivalent to 1.0 μmole of tyrosine per min at pH 7.5 at 37 °C. One protocol for that can be found at http://www.worthington-biochem.com/PROK/assay.html



    Good Luck
    David Starr


  4. Fatima,

  5. There are currently methods out there for use for of Proteinase K in antigen retrieval. My worry would be high concentration levels or excessive contact times affecting tissues you would not want to be affected. In any case here is a link I saw for some methods. FYI I don’t work for this company, I just tend to answer questions on this thread.



  7. Hi! I’m working with fecal samples and I’m trying to get DNA from Eukaryotic parasites. I have read in some articles that they use proteinase K as a really important step during the DNA extraction. May I use Proteinase K if I’m using phenol:chloroform:IAA(25:24:1, pH7.9) as part of the protocol? when should I add the proteinase K?


  9. I’m using Phusion polymerase to generate inserts with restriction enzyme sites for standard cloning and having difficulty getting the inserts in. I’m wondering if the polymerase may still be active in the digest (I run the pcr over a spin column before digesting) and filling in the ends or chewing back the ends after digestion. Has anyone used PK to remove Phusion prior to digestion with Restriction Enzymes and is there a protocol available?


  11. Ling,
    It will not damage your DNA.


  13. I would prefer if you asked the questions here. Peer review is a very powerful tool. If you promise not to be embarrassed to ask a question. I promise to not be embarrassed to say when I don’t know the answer.



    You would then discard the supernatant.
    Another spin down would occur.
    Also be sure to make sure your controls go through the same process. (Even if they don’t need it.)

    I hope this helps.

  16. Thanks David for your detailed thoughts!

  17. Vinzenz asked. “What makes you confident that even after centrifugation and lysis the/most DNA would end up in the pellet?”

    Also various types of Electrophoresis to determine quality.
    The Agilent Bio analyzer, etc.


    Good Luck



  21. Thanks David, Yeh the kit says at 56oC but I have heard varying temps for the incubation step. I ended up putting it in a -20oC freezer overnight. The reason why I put them all in ATL was because I had heard that you can leave it at that step and go back but then the book says after proteinase K digestion you can store it in buffer ATL at room temp for 6 months. Noone had told me about the ‘after proteinase K’ bit. I am going to try and borrow a massive water bath for the next samples so they can all fit, unfortunately wasn’t able to last night. I normally do it consistently and in small batches but yesterdays was quite large. Thanks again for your help :)




  23. may i know if proteinase K could degrade my DNA samples as when i’m doing my extraction, i’ve misculculate the exact amount i should put on my samples. My supervisor said, it will not give effect on my samples but then during electrophoresis, the band doesn;t seen so clear, and then, my sv said it might be due to the numerous amount of Proteinase K..is it true that it will degrade our DNA samples?

    I would think no. How concentrated are you talking about?

  25. Hi,

    1. Hi, Alba!
      Tell me, please, do you prepare this buffer by yourself?
      I tried to make the same buffer you used, and when I add SDS in the end, the solution becomes white and unsoluble clumps appear.
      I don’t understand, why it happens, couldyou please describe, step-by-step, how you make this buffer?
      Thank you!

  26. Hi,

  27. I see some issues that need to be addressed.
    The first issue is RNA interference.
    The second issue is low yield.
    The third issue is the use of Proteinase K.
    The fourth issue is the buffer.

    I would also advise including an RNAse step. RNAse is short for ribonuclease A. It will break down the RNA without affecting the DNA. I checked AG Scientifics website for it and found it priced at 0 dollars. I am going to assume by that price that they don’t have it so feel free to check out http://www.lifetechnologies.com/order/catalog/product/12091039 where I do know they do sell it. This RNAse step is usually done AFTER the break down steps and incubation but before final isolation. You might only need about 2 to 5 uL per sample for a very small time period ~ 5minutes.


    Are you advising I use a more basic buffer? Thanks!

  29. If you have a RNase step, I am puzzled why there would be RNA interference.


  31. Hello! I’m very happy to see that there is a place like that to help us in moments of almost despair LOL
    Well, lets go to the question! I’m having trouble to succeed a good extraction of some Enterococcus strains. I don’t have any kit, and I’m trying to make a home-made procedure, using CTAB detergent. Can I use Proteinase K with it? The protocol suggests that I incubate my samples with CTAB at 60ºC. Proteinase K can handle this temperature? And overnight? Or should I try SDS?


    CTAB (Cetyltrimethylammonium bromide) is a cationic surfactant that further solubilizes the cells. In general DNA is ‘happy’ in NaCl – the Na+ ions do neutralize the negatively charged phosphates on DNA and facilitate DNA molecules coming together (the molecules are less hydrophilic). A high concentration of NaCl is required otherwise CTAB-nucleic acid precipitates can form – what you are trying to do here is remove all the junk (bacterial cell wall debris, denatured proteins, polysaccharides) complexed with CTAB and leave the bacterial DNA in solution

    I don’t like reinventing the wheel so I am just going to cut and paste from another website.



  36. Oops I did some bad editing on the above post.
    Anyway good luck.


    Thanks Much. Donie

  38. Donie,



  41. Helen,

  42. hello,
    is it possible to isolate human dna from edta blood with out using prot: k…without enzyme???
    \as some of the dna isolation kits available commercially (WHICH HAS SOLUTION NAMED solu 1, slution 2, wash buffer 1 , wash buffer 2) doesnt need to be kept at -20 deg..so does that mean there is no prot: k or any other enzyme in the kit..so no need to keep it at ROOM TEMP:
    THNK U

    Also some enzymes are stable at room temp.

    Regarding my problem, I’m extracting DNA from blood by a “salting out” protocol using proteinase K. The thing is that I succesfully extract it from the blood (because of the DO 260/280 is 1.8 and I can see the DNA when I run an agarose gel) but when I use that DNA to run a PCR I can’t amplify anything. Could it be that the proteinase K isn’t working?? I am thinking about it because when I run an agarose gel with the DNA extracted from my protocol and from other protocol (the same initial sample, both) I see the “DNA band” from my protocol in a higher position in the gel, like as it was retarded by something (proteins??).


    I will take into account the RNase issue.


  48. Not in the extraction protocol, but it is the first step of the PCR protocol… I will take into account the temperature then. Thank you!




  51. 2,000 KU @ 23 KU/ul (23,000 U/ul)
    What is the concentration in mg/mL? And volume? I’m extracting from Gram negative bacteria? What is a concentration to use for a 10mL culture? Thanks!


    Thanks a lot.


  53. Shali,

    Good Luck

  54. Proteinase K is frequently frozen at -20 for long term storage. It should be stable as long as there is not too many freeze thaw cycles.

  55. Beta mercaptoethanol is a denaturing agent that affects protein structure. It does not serve as the same function as Proteinase K. It could be used in place of DTT in SDS-PAGE.
    This prevents them from digesting the RNA during its extraction procedure.

  56. I’m freeze-lysing whole blood sample with the goal of RNA recovery. I’ve been adding proteinase k after thawing the blood, but it would be ideal to freeze the blood with proteinase k, then thaw and have immediate nuclease digestion, any thoughts on freezing proteinase k? Would it still be functional?

  57. I wanted to use proteinase K to treat conditioned media. I have come across various protocols in the literature. Does anyone have experience in using proteinase K for this purpose? I am trying to find the best way to determine if the PK is working. I have treated with 200ug/ml and neutralized with 5mM Pefablock but then when using this on my cells they all died. I think the Pefablock lowered the PH of my conditioned media. I then tried using 10ug/ml followed by heat inactivation but I need to find an assay that will prove that this concentration in my conditioned media actually degraded the proteins. Any advice will be appreciated.

  58. Jeremy, I am not sure what you are asking? Are you trying to find the correct concentration levels of Proteinase K?
    I am thinking Proteinase K would kill cells if it is not inactivated. High heat has been shown to inactivate Proteinase K.
    What cell line are you using?
    Why are you neutralizing with Pefablock?
    For the Pefablock. Do a pH reading of just the media and add Pefablock to see if it affects pH.

  59. David I am using conditioned media to treat prostate cancer cell lines (PC3, C42B, PaIII, etc….) I want to use proteinase K on the conditioned media before treating the prostate cancer cells. So I would like to know what is the best concentration of proteinase K to use and the best way to inactivate it (heat or Pefablock) Thanks for any advice.

  60. Will PROTEINASE K by itself breakdown hair? If I put hair in a petri dish and added some drops of PROTEINASE K will it digest right there?

  61. Jeremy,
    I am not sure I can give you more information considering the parameters that you are doing. I am not familiar with all those cell lines. I would use heat inactivation.
    I grabbed this tidbit from the Promega Website.
    To terminate the reaction, add an inhibitor of Proteinase K such as PMSF (1) or DFP. The reaction can also be terminated by the addition of EGTA (pH 8.0) to a final concentration of 2mM or by TCA precipitation. Proteinase K may not be completely inactivated by EGTA, as this enzyme retains partial activity in the absence of calcium (7). Heat treatment (10–15 minutes at 65°C) only partially inactivates Proteinase K (inhibition by no more than 20–25%).

  62. Casey,

    I guess you can find out yourself. In theory yes, but it would take a very longtime.
    If you use heat (37 degrees Celcius), there is a better chance of it. The concentration of your Proteinase K will also affect the reaction rate.

  63. Hello Jeremy, and David, thank you both for your quick responses. We have been researching different ways of breaking down hair in the lab at school for the purpose of metals identification. This is for nutrition and or foreign substances testing. In my studies I came across a 1974 research study on Proteinase K and they touch on the subject of it breaking down human hair, finger nails, and the 1st layer of human skin. However for hair they still ended up using a pestel and mortar to crush the hairs. I was looking for a way to do this specifically with a solution. Applying heat would be acceptable. Once again thanks to both of you for your responses.


  64. It seems to me that in my DNA extraction protocol I skipped the 90C proteinase K inactivation step. I still normally spin the tissue after 2hours of 55C incubation with proteinase K buffer for 20 mins at room temperature, then I put the supernatant in fresh tubes and add cold ethanol, precipitating DNA overnight AT -20c. Then I spin the mixture and after drying off ethanol, I resuspend DNA in water. I have been having some problems with PCRs though – sometimes the PCRs would not work for me, other times only half of PCR would work. Could the lack of ProK inactivation be something that is affecting my PCR at all?

  65. Anna,
    I have seen it affect things down the line, when it was not inactivated. It can affect some things further down the line on some protocols.
    It can depend on the extraction methodology.
    The 90 degrees will also help denature for various types of protocols.

    A bunch of things I don’t see from your steps that I first think about that might affect your PCR runs.
    1. 2 Hours at 55C might not be long enough time.
    2. Your extraction method of precipitation can carry over other items. Is there a PAGE step or other steps of extraction being done?
    3. The use of RNase A to remove possible RNA interference.
    4. Normalization to make sure you are not overloading.

  66. Anna & Dave I reviewed Anna’s problem with our manufacturing lab chemist and he replied as follows ”
    It seems incredibly unlikely that the proteinase K would have any effect on the PCR reaction. After incubation at 55C for 2 hours the proteinase K would most likley be entirely degraded (by itself). If there is any active proteinase K remaining, the ethanol precipitation may denature the protein. Then, when performing the PCR, the customer only uses a small amount of the sample (thus diluting the proteinase K further). If the customer wants to be certain, they could simply incubate their sample at 95C now (to deactivate any potentially active protease), and then run the PCR reaction again.

    More than likely the customer is actually losing their DNA in the ethanol precipitation step. If the DNA concentration is very low, then it will not precipitate in ethanol, and they will lose it when they remove the supernatant.”
    Anna between David’s insights and our lab we’ll help you get this figured out. Please keep us informed if your next tests are more positive or if you continue having mixed results. – Larry

  67. Anna we have had further discussion in our manufacturing lab and these comments have come forward:
    ” The customer is doing an ethanol precipitation which will precipitate everything. Rather than an ethanol precipitation, they could purify the DNA by doing a phenol chloroform extraction, or using an affinity spin column.

    When performing PCR or quantitative PCR after ChIP, we’ve found that RNA does not contribute, and no longer bother to recommend using an RNase in our general protocol (not true for ChIP-Seq). Therefore, we would think it’s unlikely that RNA in the sample would be causing problems.

    It’s possible there are interfering factors in the sample. Rather than bother with normalization, which could be difficult if their dna concentration is very low, They could simply dilute their sample 1:10 before running the PCR to see if there’s any interference.

    I hope these suggestions are also helpful.

  68. i think that proteinase K function will be limit or decrease when SDS is mixed with proteinase K especially in case of DNa extraction from formalin preserved tissue according to my experiment and i wish to find out correct answer

  69. Sodium dodecyl sulfate is a detergent that aids in the lysing of cells and will unravel proteins for PAGE.
    I have never seen it decrease the effectiveness of Proteinase K.
    As long as you have a correct concentration level and temperature, it should work great.

  70. Due to a family emergency I had to leave my samples for DNA extraction with proteinase K @55C for 6 days (protocol was to incubate them overnight). Will the DNA still be viable for PCR or should I discard the samples?

  71. Zara,

    Although, I am sure some of the DNA would still be viable, I would expect there to be some small amount of degradation. You could run a PAGE gel to see how the bands look.
    Good science is about doing things that are repeatable, so if you deviate from your protocol, you might want to just start over. Even if the DNA is good, I would throw it out, unless it is the only sample you have to work with.
    If this is for academic purposes, you will want to document the event.
    If this is anything else besides academics, this is a deviation from the protocol and should be documented and reviewed by somebody else.

  72. Can Proteinase K be used to remove contaminates from previously extracted DNA?? I have DNA that was extracted using magnetic beads. I’d like to know if Proteinase K can be added to remove degraded DNA.


  73. Jerome,

    Proteinase K would not help in this particular situation.
    Proteinase K is normally used before magnetic bead use.
    To purify your DNA I would advise running a PAGE.

  74. But this would tie up my DNA preventing further analysis. I’m planning on running qRT-PCR but the probe isn’t binding. I think it isn’t binding because of degraded DNA inhibiting binding. I’m looking for a treatment for the DNA prior to qRT-PCR to clean it up. What are you thoughts on running it through a column??


  75. Jerome,
    Are you talking about column chromatography?
    If you are, I would say only if you have an established method already developed.
    All purification processes are going to reduce the amount of your DNA.
    Unless you know exactly what you are looking to elute off the column, you will elute the good and possible degraded DNA together.
    The PAGE can actually determine if there is an issue with your DNA if there is band separation.
    If there is band separation, you would cut the predicted band out of the gel in a light box and use that.
    You would have to remove the gel from the DNA in another step.

    I am not sure what probe binding you are talking about.
    When you say probe and qPCR, I think of the fluorescent part(The probe)of qPCR that is used during the qPCR process alongside the reverse and forward primers to indicate a successful amplification step.

    Without more information, I can not fathom a guess.

  76. My apologies for not being more explicit. By column, I mean, extraction spin columns. I’m going to try and run the sample through a filter column whereas DNA binds and junk passes through.

    As far as the qPCR, you are correct. I’m having trouble with the primers and probe binding to my target DNA.


  77. Jerome,

    I like spin columns. I don’t see harm in that.
    As for the qPCR process, there are many factors that can affect that issue.
    It would require a lot of troubleshooting considering how many factors go into the process.
    I doubt this is the best forum to address that issue.

  78. Agreed. Besides I’ve began troubleshooting the qPCR and figured purifying the DNA would be a step.

    Thanks so much for your help.

  79. Hi, I did a tail digestion O/N with an old batch of proteinase k and the tails where not well digested the next morning so I add fresh proteinase k and reincubate the tails but now I have difficulty to separate the pass during my phenol::chloroform extraction. I would like know if it could be due to an too long digestion even if I read that you can d it over the weekend or due to DNAses not well degraded by the proteinase k treatment.


  80. Helene,

    Can I get some clarification please. What do you mean by “difficulty to separate the pass”
    Also, did you crush/homogenize the tails.

    You can do digestion over the weekend, and extra contact time should not affect the quality of the DNA.
    RNA might be a different subject matter. RNA is fragile, and there are many different circumstances where RNA will degrade.
    I see you mentioned DNAses, so were you trying to eliminate the DNA after you did the extraction process to just get RNA?

  81. hi sorry, I was really tired when I write this comment. I was trying to do DNA extraction from mice tails. I did not crush the tails but digested them (100 mM tris, 10 mM EDTA, tween, NP-40 and proteinase k). Hi, I did a tail digestion O/N with an old batch of proteinase k and the tails where not well digested the next morning so I add fresh proteinase k and reincubate the tails but now after that I had difficulty to separate the phases during my phenol::chloroform extraction. I am worried that the quality of my DNA can be poor due to the old batch of proteinase k which did not degraded the DNAses enough and I was also worried to have a cloudy upper phase that never happens to me before. I did 1 more phenol::chloroform purification than usual (3 in total) to be sure that I guet read out as much as possible of the proteins that can contaminate my DNA samples.
    I hope I better explain my problem.

  82. Helene,
    I mentioned crushing the tails because that tends to give a much better digestion.
    Proteinase K is usually rather robust. I am sure the DNA you extracted was fine.
    I copied this from a paper on Phenol-chloroform extraction from Cornell.
    Typical mixtures of phenol to chloroform are 1:1 and 5:1 (v/v). At acidic pH, a 5:1 ratio results in the absence of DNA from the upper aqueous phase; whereas a 1:1 ratio, while providing maximal recovery of all RNAs, will maintain some DNA present in the upper aqueous phase.
    My guess is if the pH was acidic, and a 1:1 ration was used the DNA present in the upper phase is what is making it cloudy.

  83. Hello,
    I have a question about incubation step in extraction cell free DNA from plasma.
    Can I store the samples after 30 minutes incubation of plasma with Proteinase K and ACL buffer that contain carrier RNA and chaotropic salts for one hour?
    Do You have experience with this kind of extraction ( Qiamp circulatin nucleic acid kit )

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