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Proteinase K solution 20 mg/ml

Code
A4392

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Code & packaging Price per piece
A4392,0001
code
A4392,0001
packaging size
1 ml
price per unit
single $49,80
box price per unit
A4392,0005
code
A4392,0005
packaging size
5 ml
price per unit
single $120,60
box price per unit
A4392,0010
code
A4392,0010
packaging size
10 ml
price per unit
single $222,30
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A4392,0100
code
A4392,0100
packaging size
100 ml
price per unit
single $1996,50
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Physical Description:
Liquid
Product Code:
A4392
Product Name:
Proteinase K solution 20 mg/ml
Specifications:
DNases/RNases: not detectable
Proteinase K: min. 600 mAnsonU/ml
Composition:
Glycerol (v/v): 40 %
Proteinase K: 20 mg/mL
CaCl2 · 2H2O: 1 mM
Tris · HCl (pH 7.5): 10 mM
Hazard pictograms
  • GHS08 Hazard
WGK:
1
Storage:
2 - 8°C
Shipment:
wet ice
Signal Word:
Danger
GHS Symbols:
GHS08
H Phrases:
H334
P Phrases:
P261
P284
P304+P340
P342+P311
P501
CS:
38221900
Download TDS file for complete specifications

Comments

About Proteinase K
In molecular biology, proteinase K (EC 3.4.21.64, Protease K, Endopeptidase K, Tritirachium alkaline proteinase, Tritirachium album serine proteinase, Tritirachium album proteinase 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 can digest hair (keratin), hence the name "proteinase K". The predominant cleavage site is the peptide bond near the carboxyl group of aliphatic and aromatic amino acids with alpha-amino blocking groups. It is often used because of its broad specificity. Proteinase K belongs to the S8 (subtilisin) family of peptidases. The molecular weight of proteinase K is 28,900 daltons (28.9 kDa).

Proteinase K activity
Activated by calcium, the enzyme proteinase K digests proteins preferentially for hydrophobic amino acids (aliphatic, aromatic, and other hydrophobic amino acids). Calcium ions do not affect the activity of the enzyme but contribute to its stability. Proteins are fully digested if the incubation time is long and the protease concentration is sufficiently high. If calcium ions are removed, the stability of the enzyme is reduced, but proteolytic activity is maintained. Proteinase K has two Ca2+ binding sites, which are close to the active site, but are not directly involved in the catalytic mechanism. The residual activity is sufficient to digest proteins that normally contaminate nucleic acid preparations. Therefore, proteinase K digestion for nucleic acid purification is usually performed in the presence of EDTA (inhibition of metal ion-dependent enzymes such as nuclease).

Stability of proteinase K
Proteinase K is stable over a wide pH range (4-12), the optimum being pH 8.0. Increasing the reaction temperature from 37 °C to 50-60 °C can increase the activity several-fold, as can the addition of 0.5-1 % sodium dodecyl sulfate (SDS) or guanidinium chloride (3 M), guanidinium thiocyanate (1 M) and urea (4 M). The above conditions increase the activity of proteinase K by making its substrate cleavage sites more accessible. Temperatures above 65 °C, trichloroacetic acid (TCA) or the serine protease inhibitors AEBSF, PMSF or DFP inhibit activity. Proteinase K is not inhibited by guanidinium chloride, guanidinium thiocyanate, urea, sarcosyl, Triton X-100, Tween 20, SDS, citrate, iodoacetic acid, EDTA or other serine protease inhibitors such as Nα-tosyl-Lys chloromethylketone (TLCK) and Nα-tosyl-Phe chloromethylketone (TPCK).

Applications of proteinase K
Proteinase K is widely used in molecular biology to digest proteins and remove impurities from nucleic acid preparations. By adding proteinase K to nucleic acid preparations, nucleases that might otherwise degrade DNA or RNA during purification are rapidly inactivated. It is suitable for this application because the enzyme is active in the presence of protein-denaturing chemicals such as SDS and urea, chelating agents such as EDTA, hydrogen sulfide reagents, and trypsin or chymotrypsin inhibitors. Proteinase K is used for protein destruction in cell lysates (tissues, cell culture cells) and for nucleic acid release, as it inactivates DNases and RNases with high efficiency. Some examples of applications: Proteinase K is very useful in the purification of highly natural and undamaged DNA or RNA, since most microbial or mammalian DNases and RNases are rapidly inactivated by the enzyme, especially in the presence of 0.5-1% SDS. The activity of the enzyme toward native proteins is stimulated by denaturants such as SDS. In contrast, when measuring with peptide substrates, denaturants inhibit the enzyme. The reason for this result is that denaturants unfold protein substrates and make them more accessible to the protease. Proteinase K Inhibitors Proteinase K has two disulfide bonds, but shows increased proteolytic activity in the presence of reducing agents (e.g., 5 mM DTT), indicating that the presumed reduction of its own disulfide bonds does not lead to its irreversible inactivation. Proteinase K is inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), or 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Proteinase K activity is not affected by sulfite-modifying reagents such as para-chloromercuribenzoic acid (PCMB), N-alpha-tosyl-L-lysyl chloromethyl ketone (TLCK), or N-alpha-tosyl-l-phenylalanine chloromethyl ketone (TPCK), although it is likely to be inhibited when these reagents are used in conjunction with reducing disulfide reagents that expose the normally unavailable thiols of proteinase K.

FAQs

How is proteinase K inactivated?
Inactivation of proteinase K is perhaps one of the most common questions we receive. And the answer is very simple. Heat is a commonly used method to inactivate proteinase K. While proteinase K activity increases with temperature and is optimized at about 65 ˚C, heating at 95 ˚C for 10 minutes inactivates proteinase K. However, it should be noted that heating proteinase K does not completely inactivate the enzyme. A small amount of activity always remains with this method. Protease inhibitors such as PMSF and AEBSF (Pefabloc®) can also be used to inactivate proteinase K permanently. Note: The actual inactivation temperature is controversial and ranges from 70 to 95 ˚C. However, based on public comments and extensive research, we have decided that the best temperature for inactivation is 95 ˚C. What is the optimal temperature for proteinase K activation? As mentioned in question 1, proteinase K activity increases with temperature (to some extent). The optimal temperature for activity is between 50-65 ˚C. Higher temperatures promote protein cleavage and make it easier for proteinase K to degrade proteins. But optimizing proteinase K may not be the most important part of your process. Sometimes special techniques require adapted temperatures to achieve the best overall results. Therefore, you should also keep in mind that although the reported temperature range for proteinase K activity is wide, the enzyme is still active at temperatures between ~20-65 ˚C, and that this wide temperature flexibility can be useful for very specific methods you are performing. At temperatures above 65 ˚C, there is a risk that proteinase K will become inactive. What exactly is the relationship between proteinase K and calcium? Proteinase K binds two Ca2+ ions that help maintain the stability of the enzyme, especially when exposed to high temperatures. Calcium also protects protein K from autolysis. Calcium/heat helps maintain proteinase K's thermostability, but is not necessary for proteolytic activity. According to Richard Tullis and Harvey Rubin, this relationship becomes even more interesting when DNase I is involved. Proteinase K is known to inactivate DNases and RNases, but when DNase I is in the presence of Ca2+, it is protected by proteinase K (1 mg/mL concentration). RNase, on the other hand, is inactivated whether Ca2+ is present or not. Your results suggest a method to treat the contaminated RNase without DNaseI or to purify the highly polymerized RNA. Does EDTA inactivate proteinase K? This question also seems to come up frequently when discussing proteinase K. Chelating agents such as EDTA or EGTA have no direct effect on the enzymatic activity of proteinase K. The reason for using EDTA with proteinase K during DNA or RNA purification is usually to remove calcium (see also question #3). However, since calcium/calcium is related to the stability of proteinase K, the addition of EDTA may affect calcium and thus reduce proteinase K activity to some extent. What are proteinase K activators? Proteinase K activators are SDS (sodium dodecyl sulfate) and urea. In general, proteinase K becomes more stable and active in buffers containing these activators. How does proteinase K play a role in cell lysis? To answer this question, we must first know what proteinase K is. It is a broad-spectrum protease capable of digesting a wide range of native proteins (see below for details). In cell lysis, especially in subsequent DNA isolation and purification, proteinase K can be part of the lysis steps by digesting surface proteins. Later in the process, when it comes time to resuspend and lyse the nuclei in a buffer containing proteinase K, proteinase K contributes to the digestion of proteins that would otherwise degrade the sample. Why do many recipes for DNA extraction buffers require the use of proteinase K and RNase? This question comes up again and again because the suggestion seems like a contradiction. Proteinase K is known to digest RNases. So why put the two together in a lysis buffer? First of all, you want to add RNase because it would degrade the contaminating RNA during DNA purification. And you want to use proteinase K because it can degrade the damaging proteins, DNases and RNases. The answer to this question is a matter of timing and optimization. Some researchers suggest adding RNase first and giving it time to work. Proteinase K and SDS can then be added to degrade unwanted proteins. Some protocols require incubation of SDS, proteinase K, and RNase at 37˚C for some time. Since proteinase K activity is not as optimized at this temperature, this likely gives the RNase more time to work. The next steps in the protocol suggest a second incubation at 55 ˚C for a longer period of time, which would be an optimal temperature for proteinase K activity and allow it to digest other unwanted proteins. What applications is proteinase K used for? Proteinase K is a broad-spectrum serine protease that belongs to the class of subtilisin-like proteases (there are two types of serine proteases, chymotrypsin-like and subtilisin-like). Being a broad-spectrum protease, it is mainly used for the isolation of nucleic acids: genomic DNA, cytoplasmic RNA, high-nativity DNA and RNA, etc. It is ideal for these applications because Proteinase K is able to degrade proteins and inactivate DNases and RNases that would otherwise degrade a desired DNA or RNA sample. To simplify the list, proteinase K is used: digestion of unwanted proteins in molecular biology applications, removal of endotoxins bound to cationic proteins such as lysozyme and RNaseA, removal of nucleases for in situ hybridization, prion research related to TSEs (transmissible spongiform encephalopathies), protease footprinting, mitochthon purification, genomic DNA purification, cytoplasmic RNA purification, purification of high-nativity DNA or RNA. How should proteinase K be stored/what is the shelf life of proteinase K? Stock solution: normally stock solutions are stable at -20˚C for up to 1 year. Freeze-dried powder: normally dried at -20 ˚C for up to 2 years. However, the provisions of the respective manufacturers' certificates of analysis apply, which may differ from this statement. What can proteinase K be dissolved in / How to dissolve proteinase K? Proteinase K is very soluble in water and can also be dissolved in Tris or PBS. However, when working with PBS it can be a bit difficult, perhaps because of the pH (still in the optimal range, but at the lower end of that range). Usually, adding proteinase K powder in small amounts when shaking it in the solution will help dissolve it in the PBS. How are nuclease inactivated by proteinase K? Proteinase K is known to protect nucleic acids from protein degradation. This happens because proteinase K is able to digest proteins that would normally damage your sample. This protocol explains in more detail how to use proteinase K to inactivate nucleases during your extraction procedures. Is there an alternative to using proteinase K in DNA extractions? The advantage of using proteinase K in DNA extraction is that it can degrade a variety of harmful nucleases. It is also excellent for digesting cell membrane surface proteins. However, if this question is specifically intended to isolate DNA from other proteins, phenol-chloroform extraction is another suitable option for removing proteins from a solution. However, this method is more toxic. What does proteinase K have to do with prion diseases or TSEs? Proteinase K is involved in distinguishing between normal PrP C (prion/resistant protein) and PrPSC (disease-causing isoform). Both PrPC and PrPSC have the same molecular weight, but PrPSC is resistant to proteinase K. Samples that might contain both are treated with proteinase K, which removes PrPC and converts PrPSC to PrPRES, which has a lower molecular weight and can be detached and then distinguished. What is the molecular weight of proteinase K? The molecular weight of proteinase K is 28.5 kDa. What is the optimal pH value for proteinase K? Proteinase K is active at a pH between 7.5 and 12.0. What is the primary sequence of proteinase K? GAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGYSTIIAGMDFVASDKNNRNCPVVASLSLGGGYSVNSAAARLQSSGVMVAAGNNADARNYSPASIPSVCTVGASDRYRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA What is proteinase K? We decided to leave this question at the end, mainly because the answer is easily found on our site and is hinted at on this page. However, the question deserves its own space here. Proteinase K is a broad spectrum serine protease belonging to the subtilisin family. It is known in research for its ability to inactivate RNases and DNases that would damage desired nucleic acid samples during extraction. It is named after its originally discovered ability to hydrolyze keratin. Why is proteinase K used in DNA extraction? Proteinase K is used in DNA extraction to digest many contaminating proteins present. It also degrades nucleases that may be present during DNA extraction and protects nucleic acids from nuclease attack. What are the applications of proteinase K? Applications of next-generation sequencing (NGS) and microarray technologies: Nucleic acid purification by nuclease inactivation in DNA and RNA extraction from yeast, bacterial, mammalian cell, and plant cell lysates, improving cloning efficiency of PCR products, Sample preparation for quantification of DNA adduct levels by accelerator mass spectrometry, inactivation of enzyme cocktails in ribonuclease protection assays, addition to extraction procedures to optimize RNA yield from primary breast tumors for microarray studies. Molecular biology applications: detection of bovine spongiform encephalopathy proteins that show unique resistance to proteolytic degradation. Tissue digestion (protein denaturation) as an alternative sample preparation for quantitative analysis by liquid chromatography-tandem mass spectrometry. Specific modification of cell surface proteins to analyze membrane structures for protein localization, generation of protein fragments for characterization of functional studies. What are the guidelines for the use of proteinase K? Isolation of high molecular weight DNA: Chromosomal DNA that has been embedded in agarose plugs can be treated with proteinase K to inactivate the restriction enzymes used to digest the DNA. The enzyme is used for this method at a concentration of 1 mg/mL in a buffer containing 0.5 M EDTA and 1% N-lauroylsarcosine (v/v). Incubate for 24-48 hours at 37 °C. Isolation of genomic and plasmid DNA: Genomic or plasmid DNA can be purified from cells frozen in liquid nitrogen or from cultured cells using proteinase K. Incubate 50-100 mg of tissue or 1x108 cells in 1 ml of buffer containing 0.5% SDS (w/v) with proteinase K at a concentration of 1 mg/mL at 50 °C for 12-18 hours. RNA Isolation: For cytoplasmic RNA isolation, centrifuge the cell lysate, remove the supernatant, and add 200 ug/mL proteinase K and 2% (w/v) SDS. Incubate for 30 min at 37 °C. Total RNA can be isolated by separating the lysate before enzyme treatment and is extracted through a needle attached to a syringe. Inactivation of RNases, DNases, and enzymes in reactions: proteinase K is active in a variety of buffers. The enzyme should be used in a ratio of approximately 1:50 (w/w, proteinase K: enzyme). Incubate at 37 °C for 30 minutes.Why does digestion occur at 50 °C? By increasing the temperature to 50 °C, some proteins are unfolded so that they can be degraded more easily by proteinase K. The enzyme is stable and its activity increases. The enzyme is stable and its activity increases considerably when denaturants such as SDS and urea are added. What is the fastest and most efficient way to inactivate proteinase K? As with most proteins, the most efficient way to inactivate the enzyme is to increase the temperature or significantly change the pH. Proteinase K is inactivated by heat (e.g., incubation at 55°C). How can you tell if the enzyme is working? To determine if the enzyme is working, you can perform the following 2 steps: Determine how many micromoles of p-nitroanilide are produced per minute. Then divide by the total amount of protein in the solution. This way you can determine the specific activity of the enzyme = units (one unit equals 1 mole of p-nitroanilide produced per minute), specific activity = units of enzyme activity/mg of total protein. Where is proteinase K cleaved? Proteinase K cleaves peptide bonds plus the carboxyl group of hydrophobic, N-substituted aliphatic and aromatic amino acids. It also cleaves peptide amides. What is the lifetime of proteinase K? Proteinase K has a shelf life of 12 months when stored in a dry place at 4-8 °C, as it is very stable. Short-term storage at room temperature will not affect the activity and stability of proteinase K. However, the manufacturer's conditions in the certificates of analysis apply. What is the role of proteinase K in the Covid-19 assay? Proteinase K plays an important role in the preparation of samples for the PCR assay for Covid-19. The function of proteinase K is the digestion of proteins in the sample, particularly nuclease, which would otherwise promote the degradation of DNA and RNA in the sample and thus distort the final test result. Where is proteinase found in nature? Proteinase was first discovered in 1974 in extracts of the fungus Engyodontium album (formerly Tritirachium album).

Literature

[1] Betzel C, Singh TP, Visanji M, Peters K, Fittkau S, Saenger W, Wilson KS (July 1993). "Structure of a proteinase K complex with a hexapeptide inhibitor analogous to the substrate at 2,2-A resolution". J. Biol. Chem. 268 (21): 15854-8. [2] Morihara K, Tsuzuki H (1975). "Specificity of proteinase K from Tritirachium album Limber for synthetic peptides". Agriculture. Biol. Chem. 39 (7): 1489-1492. [3] Kraus E, Kiltz HH, Femfert UF (February 1976). "The specificity of proteinase K against oxidized insulin B-chain". Hoppe-Seyler Z. Physiol. Chem. 357 (2): 233-7. [4] Jany KD, Lederer G, Mayer B (1986). "Amino acid sequence of proteinase K from the mold Tritirachium album Limber". FEBS Lett. 199 (2): 139-144. [5] Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H (August 1974). "Proteinase K from Tritirachium album Limber". Eur. J. Biochem. 47 (1): 91-7. [6] Müller A, Hinrichs W, Wolf WM, Saenger W (September 1994). "Crystal structure of calcium-free proteinase K at 1.5 A resolution". J. Biol. Chem. 269 (37): 23108-11. [7] Hilz H, Wiegers U, Adamietz P (1975). "Stimulation of proteinase K action by denaturants: application to nucleic acid isolation and degradation of 'masked' proteins". European Journal of Biochemistry. 56 (1): 103-108. [8] Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. & Struhl, K. (eds.) (1995) Current Protocols in Molecular Biology. Greene Publishing & Wiley-Interscience, New York [9] Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition page B16. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. [10] Müller, A. et al. (1994) J. Biol. Chem. 269, 23108-23111 Crystal structure of free Calcium-Proteinase K at 1.5 A resolution. [11] Wallace, D.M. (1987) Enzymol Methods. 152, 41-48 Small and large scale phenol extraction. [11] Breyer, J., Wemheuer, W. M., Wrede, A., Graham, C., Benestad, S. L., Brenig, B., . Schulz-Schaeffer, W. J. (2012). Detergents alter PrPSc proteinase K resistance in several transmissible spongiform encephalopathies (TSEs). Veterinary Microbiology, 157(1-2), 23-31. [12] Charette, S. J., & Cosson, P. (2004, September). Rapid preparation of genomic DNA for PCR analysis. Tullis, R. H., & Rubin, H. (1980). Calcium protects DNase I from proteinase K: a new method for removal of RNase contaminating DNase I. Analytical Biochemistry, 107(1), 260-264. [13] Valeria Genoud, Martin Stortz, Ariel Waisman, Bruno G. Berardino, Paula Verneri, Virginia Dansey, Melina Salvatori, Federico Remes Lenicov, Valeria Levi (2021) Non-extraction protocol combining proteinase K and thermal inactivation for the detection of SARS-CoV-2 by RT-qPCR. https://doi.org/10.1371/journal.pone.0247792