10 Questions You Want To Ask About Proteinase K


10 Questions You Want To Ask About Proteinase K

Do you need Proteinase K? We’re your source! Learn more at AG Scientific!

What is Proteinase K?

In molecular biology Proteinase K (also known as protease K or endopeptidase K) is a broad-spectrum serine protease. The enzyme was discovered in 1974 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).

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.

    AG Logo with test tubes
    Photo by Sarah Pflug from Burst

    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., 2008), 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.

    DNA strands

    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?

    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

    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.

    Proteinase K Protein Structure




    • 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.
    • 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.
    • Miller A, Hinrichs W, Wolf WM, Saenger W (1994). “Crystal structure of calcium-free proteinase K at 1.5 resolution”. The Journal of Biological Chemistry 269: 23108-23111. PMID 8083213.
    • http://wiki.answers.com/Q/What_is_the_function_of_Proteinase_K_in_DNA_extraction
    • Wikipedia.

161 thoughts on “10 Questions You Want To Ask About Proteinase K”

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    1. Hi Jeremy –

      Thank you for your comment. Glad to hear that you are enjoying our content.

      Happy reading!

      Amrutha Karuturi
      Digital content Manager
      AG Scientific

  2. Really useful content! Interesting to see Prot K can be shipped at ambient temperature. How long can Proteinase K be kept at room temperature? Thanks!

    1. Hi Anita,

      We highly recommend storing PK at 4 degrees Celsius if you plan for long-term storage, but leaving PK out for a few days at room temperature should not ruin the reagent.

      Thanks for commenting!

      Trisha Timpug
      Co-Director of Marketing and Sales
      AG Scientific

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    1. Hello Jacki –

      Thank you for the positive feedback. We have talented writers on staff, so I will pass the message along. Glad that we stand out from the other blogs that you are reading.

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      Idelle Delapena
      Marketing Director
      AG Scientific

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    1. https://www.researchgate.net/post/How_can_I_chemically_inactivate_Qiagen_protease_prior_to_DNase_treatment

      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 ËšC, heating proteinase K to 95 ËšC 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 ËšC. However, crowd sourced feedback and extensive research led us to settle on 95 ËšC as the best temperature for inactivation.

      Hope above information helps you.

      Thank you,

      Director of Web Content

  5. I forgot to put my proteinase K back into the fridge last night after using it. it has been at 20C for about 16 hours. Will it still be active?

    1. Hi Kate – Yes it will still be active. Proteinase K can actually be shipped at ambient temperature. If you have other questions, feel free to email me directly at idelle (at) agscientific (dot) com.

      Thank you for reading!

      Idelle Delapena
      Marketing Director
      AG Scientific

  6. Hi all.. am now onsite trying to extract rna from blood .. the issue that I don’t have ddt or percipta .. cani use proteinase k instead. . Or to heat 60 c for 10 min .. or any advice… thnx

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  22. Hi,

    I have a question about the inactivation step for Proteinase K.
    Our protocol for DNA extraction from tail biopsies requires 10 min inactivation at 95 degrees. My samples were almost an hour at 95 degrees. Would this affect the integrity of DNA? Is there anybody experienced problems due to prolonged inactivation time?


  23. Hi,

    Just wondering if anyone has ever come across a case where the ProK appears to be eating the DNA?
    We often get blobs at the bottom of the gel after a DNA extraction, so wanted to test the ProK. We added 5 different samples of ProK to DNA which did not have the big blob present, and incubated at 55 degrees for about 5 hours. A control of the DNA sample without the ProK was also subjected to the hot block and subsequent spinning in the centrifuge.
    When run on a gel the only DNA that was still visible was that which had not been treated by ProK. Any thoughts on why this occurred????


  24. 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 )

  25. 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.

  26. 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.

  27. 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?

  28. 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.


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

    Thanks so much for your help.

  30. 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.

  31. 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.


  32. 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.

  33. 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??


  34. 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.

  35. 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.


  36. 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.

  37. 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?

  38. 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.

  39. 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

  40. 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.

  41. 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

  42. 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.

  43. 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?

  44. 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.


  45. 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.

  46. 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%).

  47. 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?

  48. 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.

  49. 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.

  50. 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.

  51. 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?

  52. 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.
    2-Mercaptoethanol is used in some RNA isolation procedures to eliminate ribonuclease released during cell lysis.
    Numerous disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins.
    This prevents them from digesting the RNA during its extraction procedure.

  53. 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.

  54. Shali,
    I cannot speak for the validity of the claim by your boss of 10 year old Proteinase K being viable. Unless there is some type of proven stability study by the manufacturer, I would assume an enzyme would lose effectiveness over time, even if lyophilized.
    I am used to a good manufacturing environment with regulations governing expiration dates. Laboratories that do work in regulated environments, will usually toss any chemical or consumable that is not a sample after 5 years. Otherwise an auditor is going to want to see some stability studies, and if none exist you are going to be facing some sever scrutiny. Even most academic environments have expiration dates.
    That being said, you can still test the Proteinase K on other types of samples to see how effective it is compared to brand new Proteinase K.
    My experience with mice tails usually meant I had to crush the tails into a pulp before adding my lysing buffers and Proteinase K. Otherwise I did not have measurable results. Bone and cartilage are not very easy to extract DNA from. Sometimes a more robust extraction buffer is used, as well as longer heat times.
    Without actually seeing the measurement graph of your 50ng/µL I cannot determine if you are having RNA interference or a blanking issue. To reduce RNA interference I would advise an RNAse step.
    In the past I rarely resolved DNA into water. Usually, I have left the DNA in the extraction solution, and I have used a sample of the extraction solution as a blank.

    Good Luck

  55. Hi, I have a question about Proteinase K. I am doing some genotypes on mice. When I extract DNA from tail, I always see dark color DNA, After PCR, I run the gel, I could not get the band. I used the Proteinase K that were purchase 10 years ago. My boss said it should no problem. But I try several times, there are still no bands and every time I got the card colour DNA. These kind of DNA can be resolved in nano pure water. when I measure the concentration of DNA, I can have around 50ng/ul. I think my Proteinase K might lose its activity. Any suggestions?

    Thanks a lot.


  56. 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!

  57. I need to add 100ng/ul of lysozyme, but my stock enzyme is in U/ul. I have never worked with this unit. Can you explain to me how to convert from U/ul to ng/ul?


  58. Due to an error, a shipment of lyophilized Proteinase K was left at room temperature (~20 C) for 3 days.
    I am planning to use it soon (in the next two months), but am wondering if you think the enzyme will still be effective? I tend to think it will be OK since it was lyophilized and not in a very hot situation, but would like another opinion. Thank you!

  59. 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!

  60. Agustin, can be deactivated at that temperature. If you read the top of the page on question 5. They indicate that Proteinase K Proteinase K is inactivated by heat, eg. Incubating at >55 °C. I have used it at higher temperatures myself, but that was because it was in large amounts and it was a short time period. I do not know who manufactured your kit. I would think they would have tested everything. Sometimes a kit is not perfect for an end users needs. Maybe the maker did not take in account for gel work. That is why I suggested the test with the four samples. It is to help figure out if the problem is temperature related or RNA interference. Proteinase K would not have an affect on the TAQ polymerase if it was deactivated. Is there a high heat step to bring the sample to 95 degrees for say 10 minutes to completely inactivate the Proteinase K?

  61. Thank you very much David, but 65ºC is the temperature recommended in the data sheet of the protein. Should I try less temperature?? Could it be possible that I am not removing completelly the Proteinase K and it would be inactivate
    ing the Taq polimerase when I add it to start with the PCR??
    I will take into account the RNase issue.

  62. The 65 degrees is a high end for Proteinase K and can actually be inactivated at a temperature greater than 55ºC. So that might be too high. (It doesn’t always happen quickly at that temperature). There is also a possibility that RNA is interfering as well. If there is not an RNAse A step to eliminate RNA, interference might occur. The only way to find out is to do an experiment. 4 samples. Two sample as done by the protocol and one of them with an RNase A step. Run a 2nd set of samples with the same protocol but at 55ºC and with one of the samples with an RNase A step.

  63. Hi! I ve found this page looking for a solution to my problem. It seems very useful, thank you for share you knowledge.
    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??).
    The digestion step of the protocol is at 65ºC. Is that too much??
    Thank you in advance and i beg you apologies for my bad English. I hope it is clear enough

  64. Yes to the first question. It is possible to isolate human DNA from edta treated blood without using Proteinase K.
    I don’t know what are ingredients in any of the solutions. so I rather not make assumptions that the manufacturer should be able to answer.
    Also some enzymes are stable at room temp.

  65. 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

  66. Helen,
    It is a simple question. Your question is actually very complicated to answer economically, and leads to many questions. If there is any form of GMP involved in your operations, you have to weigh in costs of time, labor, validation, SOP writing, QC, QA, equipment usage and cost. There will also need to be stability studies to determine expiration dates. Your lots will need to be tested before release for use for various profiles and potency to determine, if your substance is going to be usable in a GLP or GMP environment. What type of QC tests will you use? Endonuclease and Exonuclease Activity? Non Specific and specific DNase activity? Contamination screening? RNase activity? HPLC profiling? PAGE testing? Mass Spec? Of course the usual paperwork will be involved. MSDS, certifications, lot validation..etc.
    If you are strictly a research facility that does not care about quality control and has no ties to any form of cGMP or industry standards, than maybe. You still need to invest in the time, and resources. Consider the labor expense in the manufacturing. Also consider that you still want the Proteinase K to be free of contamination. How much is your labors time worth, and what are your deadlines? How qualified is your labor source to manufacture the Proteinase K? Will your production of Proteinase K be a variable in the successful repeating of your scientific experiments? That variable of repeatability might make it not worth it.
    If you are in a strictly academic training environment, where there are no consequences to research and the labor is cheap. I think it could be worth it. Even then, I would advise buying Proteinase K to test against the manufactured lots for a comparison standard.
    There are still other questions to ask and answer. I don’t know of an expression system to manufacture Proteinase K per se. Proteinase K is extracted from the fungus Engyodontium album (Tritirachium album Limber) which grows on Keratin and some other substances. Will you grow your own Engyodontium album ? What incubation times will you use? Will you use a fermentation process? What type of fermentation? Will you use conventional submerged fermentation? Will you use SSF (Solid state substrate Fermentation)? What will you grow your fungus with on a substrate? What type of substrate, grain or bran? What are your incubation times; 37 degrees for 48 hours or 25 degrees for 120 hours? How will you optimize your pH? What nutrients will you add for optimization? Will you extract using distilled water slurry? How will you separate the slurry? Centrifuge filtration? Gravity separation and filtration? Will you use ammonium sulphate precipitation? Will you want to purify it? You might need some form of chromatography and electrophoresis combination to purify. Once it is made in bulk how will you store it? What type of storage buffer will you use? Tris base buffers with 50%glycol? What temperature? -20 for long term for up to 2 years? Will you aliquot it first into smaller aliquots to prevent freeze thawing of the entire lot?

  67. quick question, in order to cut-down costs is proteinase K worth making on our own? how successful can we be? which expression system works best? Thanks alot.

  68. Could somebody help me with the best conditiond for Proteinase K to autodigest in the process of digesting other proteins in the solution. Basic aim is the have a final solution with least protein remaining. Thanks.

  69. Donie,
    Lyticase is normally used for the isolation of DNA from yeast and fungus, so given the choice of using Proteinase K or Lyticase on bacteria I would use the Proteinase K.
    I am not sure what you are asking about 16SrDNA. The S in 16 S stands for Svedburg unit and is the number is based on sedimentation rates of a particle, with the larger numbers settling faster. rDNA is a DNA sequence that codes for ribosomal RNA. Ribsomes are organelles and are usually the primary site for biological protein synthesis in cells. Since species and strains of bacteria usually need very specific proteins for their functioning, we can usually find strands of DNA very specific to a species in ribosomal DNA.
    18s rRNA is found in eukaryotic DNA and its counterpart 16s rRNA is found in Prokaryotes and mitochondria. So a test for 16s rDNA using specific primers and probes, DNA isolation, amplification, sequencing, and electrophoresis can create a specific test to identify Prokaryotic bacteria strains and species against a known data base.
    If you would like to check out a good protocol and test for 16s rDNA please check out
    Go down to manuals and products and feel free to read the pdf files for the manual and protocol to get a better understand of the process and procedures involved.

  70. Hello, and thanks for the information that is posted. I would like to find out what protein should be used to lyse the bacterial cell in DNA isolation? would it be Proteinase K or lyticase? and secondly please explain to me what is 16SrDNA and how it works?

    Thanks Much. Donie

  71. Oops I did some bad editing on the above post.
    I was trying to ask if you would use NaOH with the SDS.
    Anyway good luck.

  72. I am sure it worked to a point, but that does not mean it worked effectively to be usable for any sort of provable scientific data.
    PCR has various heating and cooling steps. IF the power went out during one of the stages where high heat was needed, your cycle will have issues. When the temperature dropped from the extension/elongation phase it would have most likely dropped into an annealing phase and than dropped to a colder level.
    Even if everything was perfect, I would never use that data.

  73. There is a powercut of 20minutes in the University today and I had a PCR reaction running. The reaction resumed after 20minutes, I am wondering if the PCR going to work?

  74. As far as I Know Proteinase K can handle 60 degrees but 55 tends to be ideal and higher temperatures it is not as effective.
    CTAB Cetrimonium bromide is a Cationic surfactant. SDS Sodium dodecylsulfate is an anionic surfactant. Your bacteHydroxide with the SDS? Is Chloroform involved? Honestly, unless I saw the whole protocol, I would not even know where to began. Proteinase K might not be needed if using the NaOH. It doesn’t hurt, but you are just breaking down bacterial strains. do you need the cells completely broken down and lysed?
    ria is a gram positive bacteria and has a single lipid bilayer. Enterococcus is also very tolerant of high sodium environments and up to 45 degrees celcius.
    Are you using Sodium Hydroxide? Are you using a chlorophyl or bead method?
    I don’t like reinventing the wheel so I am just going to cut and paste from another website.

  75. SDS, an anionic surfactant (often found in many cleaning products like hair shampoo), does lyse the bacteria. In alkaline lysis plasmid DNA minipreps SDS is often used in conjunction with NaOH (e.g., 0.2N) following a lysozyme enzyme digestion, and then you would not typically have a proteinase K step. PK is more often used with genomic DNA isolation (without alkaline lysis) or even after a restriction enzyme digest. So proteinase K will inactivate nucleases (e.g., DNases/RNases) particularly in the presence of SDS, but it a broad-spectrum protease that also digests proteins to ensure complete lysis of the cells.

    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

  76. 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?

  77. The second portion of the protocol is a kit that comes with RNase A in it. Instead of another phenol:chloroform step, a kit is being used to removed the remaining debri after RNase A incubation step. Thank you very much for the feedback!

  78. If you have a RNase step, I am puzzled why there would be RNA interference.
    Most kits are set up with certain conditions that have most likely been optimized by the manufacture of the kit. At this point, I have to wonder if you are using a Kit. If you are using a Kit, I would think calling the manufactuers customer service is the best bet. IF not, then yes I would raise the pH to the 8.5 to 9 range.

  79. Thanks for the feedback! So my protocol does have a RNase A incubation at 37 C for 20 minutes, step after the phenol:chloroform step.

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

  80. 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.

    About that RNA interference; Although, I think your buffer pH might be better between 8.5 to 9 pH for the Phenol:chloroform extraction. This is based on experience. I have read how pH in chloroform extraction can be adjusted to separate DNA from RNA, but I am not an expert on that. I will steal these words from Wikipedia.
    In brief, aqueous samples are mixed with equal volumes of a phenol:chloroform mixture. The proteins will partition into the organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the aqueous phase. If the mixture is acidic, DNA partitions into the organic phase while RNA remains in the aqueous phase.
    This is why I would advise a more basic pH, you might be losing some DNA.
    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.
    The lysing buffer is designed to denature the proteins and break up the cellular walls to allow for a release of the DNA and other material in the targeted material. Proteinase K will not harm your DNA and can be added with the lysing buffer. To increase your DNA yield you could increase your buffer/Proteinase K contact time with the target. The higher the temperature and contact time with Proteinase K, the higher the effectiveness.

  81. Hi,
    I am trying to isolate genomic DNA from green algae called Chlamydomonas. I was given the protocol to try it put but I have a problem with low yield and RNA in my sample. The protocol has a Lydia buffer consisting of nuclease free water, Tris/HCl pH 8, NaCl, EDTA, 2% SDS, Qiagen
    Proteinase K, and PVP 1%. I am using cell pellets
    that are frozen on try ice and each cell pellet is equal to 1.5 X 10^8. One 1mL of lysing buffer is used for each pellet and pipetting 12 times is used to dissolve the pellet. The phenol:chloroform ph 7.9 is used and incubate at room temperature for 5 minutes the spin for 5 minutes. My question is if it’s okay to used proteinase K in this lysing buffer. Can you suggest a better order to used proteins K or a better lysing buffer.

  82. Hi,
    I am trying to isolate genomic DNA from green algae called Chlamydomonas. I was given the protocol to try it put but I have a problem with low yield and RNA in my sample. The protocol has a Lydia buffer consisting of nuclease free water, Tris/HCl pH 8, NaCl, EDTA, 2% SDS, Qiagen
    Proteinase K, and PVP 1%. I am using cell pellets
    that are frozen on try ice and each cell pellet is equal to 1.5 X 10^8. One 1mL of lysing buffer is used for each pellet and pipetting 12 times is used to dissolve the pellet. The phenol:chloroform ph 7.9 is used and incubate at room temperature for 5 minutes the spin for 5 minutes. My question is if it’s okay to used proteinase K in this lysing buffer. Can you suggest a better order to used proteins K or a better lysing buffer.

    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!

  83. On one of my jobs in the past somebody added 10x the amount of Proteinase K than they should with no ill effect to the sample.
    I would think no. How concentrated are you talking about?
    I doubt the Proteinase K might affect the acrylamide gel you are using in your electrophoresis.
    I guess you could experiment with that by running various concentrations of Proteinase K with the marker through a gel.
    Do you have any other way to test your sample? Nano drop? Spec? Agilent Bio Analzyer? etc

  84. 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?

  85. No problem. In the past I have usually ran the proteinase K assay at 55 degrees or 37 degrees celcius. The 37 degrees was usually for over the weekend digestion while the 55 degrees was usually for shorter digestions.

    I have extracted DNA out of samples that have been frozen at -20 AFTER the Pro K digestion step.
    It is fine, but it might not be good science. I have never had samples sit at room temperature with Pro K in them for an extended period of time. Keeping things at room temperature can allow other organisms to grow in your samples.

    The thing is you want to have a good control with defined stop steps. So you might want to avoid situations where the control does not go through the same steps as the samples.

  86. 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 🙂

  87. The Proteinase K is much more effective at the 55 degrees setting. ATL contains detergents that will help unfold the proteins allowing the Proteinase K to be more effective. The ATL should not degrade your DNA. Room temperature on the other hand could help certain organisms possibly replicate in your sample. So cold storage or freezing might be a good idea. You should be able to store the samples with the ATL in the cold and add the Proteinase K later. I would suggest -20 Freezer for long periods of time.
    I don’t know what your samples are being used for, but it is good science to keep certains steps consistant with each other. This way you can keep your control samples to a minimum.

  88. I was just wondering if anyone could help me, hopefully I will get it in time as I left this question last minute. I know with the Qiagen DNeasy kit you put buffer ATL in with your tissue samples and then add proteinase K and then incubate at 56oC. I have however added buffer ATL already to my tissues then realised I don’t have enough room in my waterbath for all my samples. Is it ok to leave the tissues in buffer ATL for a day or 2 when I can fit them all in etc? Or should I add proteinase K to them, vortex and then just store it at room temp? OR freezer even? Any help would be much appreciated, Thanks!!!

  89. I wish I had a better option for you with the 10,000 plus samples you have to deal with, but most clots are going to need some type of serious agitation to be broken up. Heat sources can add energy to the process and reduce the agitation needed, but at the same time that might affect the DNA quality.

    Simple up and down automated pipeting has not worked well for me in the past and the tips could easily get clogged. I also have no idea what type of extraction/isolation method you are going to use on the eluent and the time constraints.
    Good Luck

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

    All I can say is experience and the testing of the Optical Density (OD) of the DNA versus a blank of the eluent solution.
    Also various types of Electrophoresis to determine quality.
    The Agilent Bio analyzer, etc.
    There are various types of extraction/isolation methods out there, I am just trying to help you figure out how to deal with the blood clots and if and when to use the Proteinase K.

  91. Thanks David for your detailed thoughts!
    I am a little surprised that you suggest to discard the supernatant after the first spin and even after proteinase K treatment. I would have thought that after extended storage times many cells would have been lysed and a large fraction of the DNA would end up in solution/supernatant. What makes you confident that even after centrifugation and lysis the/most DNA would end up in the pellet?

  92. I am not sure how you are doing the isolation. Some methods vary. Although Proteinase K would definently be helpful, it could be done during or after the Lysis and wash steps. Just remember Proteinase K usually works better in above room temperature applications. There are various lysis buffers as well. I am not sure which you are using.
    In most cases you would take the blood samples out of the cold and if frozen leave them out over night to thaw. This will also help the blood cells to lyse.

    Normally if blood has not been stored for a long period of time or frozen, pipet agitation can be used, but in this case the clotting can be broken up with vortexing. Considering the storage time, I would think that would be best. Sorry, you are going to have to do manual labor to get the best results.
    Make sure the tubes holding the blood are well sealed and have no cracks.
    Inside a a biosaftey cabinet or hood place a vortexer and individually place the tubes on the vortexer one or two at a time and try to break up the clots.
    You will then want to do a spin down 5 to 10 minutes at around a few thousand rpms.
    You would then discard the supernatant.
    At this point you might do a lysis buffer step. You could add a certain amount of lysis buffer with protenase K and cap the tube and vortex like crazy to break up the pellet and clots. An incubation period can occur at this step.
    Another spin down would occur.
    The supernatant would get discarded and then you can do your wash and elution steps.
    Also be sure to make sure your controls go through the same process. (Even if they don’t need it.)
    If you look up ChargeSwitch® gDNA Purification Kits from Life Technologies you can see a protocol where they use Proteinase K with the Lysis buffer and it is done at room temperature. You can look around the net for other blood with proteinase K extraction or isolation methods. I hope this helps.

    I hope this helps.

  93. We are supposed to isolate DNA from full blood samples that have been stored for several years. Despite anticoagulants most samples are clotted. How could we prepare those samples for automated pipetting (>10.000 samples). Would proteinase K work? Other enzymes?

  94. 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.
    If this is a proprietary issue, I might not be the correct person to ask.

  95. Oh thanks for the prompt reply. Hopefully it is not gonna give a big difference like you said. Are you in the line of molecular biology?

  96. Ling,
    Chelex is used to protect the DNA from the heating process that is used with Proteinase K. It does with a binging of Mg2+ ions during extraction.
    It will not damage your DNA.
    10 minutes of time should not affect your DNA purity at all.
    This is assuming everything was done at room temperature. If you were extracting for RNA, a time crunch might be an issue.

  97. I am doing DNA extraction with chelex with eel tissue.The protocol is to add Proteinase K right after adding the chelex. However, today I waited for more than 10 minutes after the chelex before I added Proteinase K. Is it going to affect the purity of the DNA?

  98. 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?

  99. Proteinase K is used in DNA extraction as it digests proteins especially nucleases that may degrade DNA/RNA. Activity of protienase K is enhanced in presence of SDS. Proteinase K can be used with phenol:chloroform:IAA protocol and it is generally added after the cell lysis step. The cell lysate to which proteinase K has been added is then incubated for 2-3 hours. The time and temperature for Proteinase K activity might have to be optimized. -Pratima Doshi, Guest Blogger

  100. 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?

  101. think you all, but I have a question. Would this proteinase K used as Antigen Retrival for anti-body specific binding?

  102. 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.

    I think the method 2 might be better. If it was a learning process and your tissues are valuable I would be cautions and go with lower temperatures and contact times and adjust upwards until you find a happy medium.

  103. Fatima,
    I do not understand what you mean by a weak band in pcr product. To clarify, does this mean you are doing PCR on your extraction and then taking the replicated DNA and separating it with a gel to get a band?
    It would also help to know what tissue you are extracting the DNA from and the method you are using to extract the DNA. In most cases the proteinase K is usually used on the tissues BEFORE the DNA extraction process. Frequently it will be a digestion process between 37 to 55 degrees from 1 hour to overnight or over weekend time periods. This mixture then might be heated to 95 degrees for a small period of time to inactivate the Proteianse K. This digestion process may be done with various detergents added to allow for proper digestion of any proteins. Some of those detergents may also cause interference. I have read of SDS interference being inactivated by Tween 20. As for your primers, I am sure they are fine. Your Master Mix might be an issue. How sensitive is your master mix to temperature changes? What is the contents of the master mix. Perhaps switching out dTTp with dUTP and using UNG will help with any amplicon carry over and false positives.

  104. hello.i have a problem in my DNA extraction, and have weak band in pcr product,now i want to use protein kinase k to remove proteins,i wanna know that`s right way? ichecked primer and other material.any suggestion??? tnx alot.

  105. To Maria Romero.
    If you have any albumin or some other protein, you can use the Proteinase K on the Albumin first and run a gel comparing the Proteinase K + Albumin versus a control of just Albumin and a control of just Proteinase K.

    If the Albumin was digested you should get plenty of bands in your gel.
    Proteinase K will also cleave benzoyl arginine-p-nitroanilide, which gives you a yellow p-nitroaniline. This can be detected at 410 nm on a spectrophotometer. This is an activity test that is sometimes done for varius enyzymes.

    You can compare activity of your Proteinase K over time with the spectrophotometer method to check the activity.

    You can compare the Proteinase K on the day you prepped(opened) it, versus the next time you use it.

    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

    You could also call up the manufacturer and ask them what does the COA says and find out how they determine activity.

    Your Proteinase K activity could have been inhibited by EDTA, DIFP, or PMSF. So you might want to check for those type of containments.
    If the pH was very low or if the temperature range was too high or too low, that could have been an inhibitory issue as well.

    Good Luck
    David Starr

  106. Hello everybody I just found your website, which I find very useful! I have a concern regarding proteinase K, I normally use it at 10micrograms per ml of PBS and works perfect at the moment to perform insitu hybidization in fish, however last time I prepared more and it failed since my fish did not present any staining. I did noticed that 3 days after I have started my insitu for that reason I would like to know wheter there is a method to test proteinase K works before using it. Kind regards Maria Romero

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