Proteinase K Dna Extraction Method
Proteinase K is one of the best DNA extraction methods to date. The method is accurate, reliable and fast. The proteinase K DNA extraction method is also called as an enzymatic method of DNA extraction because of the important role of proteinase K enzyme in the DNA extraction.
In this article, we will discuss the DNA extraction by proteinase K enzyme. The major topics of the article are:
Size Aggregates In Scjd
PrPSc shows three distinct patterns of sedimentation, before and after PK treatment . In MV1 sCJD subtype, PrP is composed by soluble forms of small size and by large insoluble aggregates, before and after PK treatment. In MV2 sCJD subtype, PrP is widely distributed all over the gradient, while after PK digestion PrPSc is detected mainly at the bottom of the gradient as insoluble aggregates of large size. Disease-associated PrP in MV-U sCJD subtype shows a sedimentation pattern sharing properties with both PrPSc Types 1 and 2.
Fig. 14. Fractionation of sCJD PrP aggregates. Brain homogenates from frontal cortexes of MV-1 and MV-2 and MV-U were sedimented in a 10% to 60% sucrose gradient. After sedimentation, half samples were digested with PK. Relative cellular and PK-resistant percentage of fraction.
Application Of Proteinase K Dna Extraction Method
The proteinase K DNA extraction method is one of the most successful DNA extraction methods reported to date. It is the first choice for genomic DNA extraction for microarray and sequencing.
The DNA obtained from the proteinase K method is highly pure and the quantity of DNA is also very good. Further, the method is simple and rapid.
It is also used in mRNA extraction. It is very effective for deactivation of DNase and RNase.
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Applications For Next Generation Sequencing And Microarray Technologies:
- Nucleic acid purification by inactivating nucleases when extracting DNA and RNA from yeast, bacteria, and mammalian cell & plant cell lysates
- Improves 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
- Added to extraction procedures to optimize RNA yields from primary breast tumors for microarray studies
Why Do Many Dna Extraction Lysis Buffer Recipes Call For Proteinase K And Rnase
This question comes up every now and then because the suggestion seems like a contradiction. Proteinase K is known to digest RNases, so why would the two be added together in a lysis buffer? First of all, you want the RNase added because it would break down contaminating RNA during your DNA isolation. And you want to use proteinase K because it will break down damaging proteins, DNases and RNases.
The answer to this question is really rooted in timing and optimization. Some researchers suggest adding the RNase in first, allowing time for it to work. Then proteinase K and SDS can be added to break down unwanted proteins. Something else to consider is that some protocols will have you incubate in SDS, proteinase K and RNase at 37 C for a certain amount of time. Since proteinase K activity is not as highly optimized at this temperature, this likely gives RNase time to work. Later steps in the protocol suggest a second incubation at 55 C for a longer period of time, which would be a more optimal temperature for proteinase K activity, allowing it to digest other unwanted proteins.
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Why Is Knowing The Activity Of Proteinase K Important
We actually dedicated a whole article to explaining more about proteinase K activity and concentration. The scenario we presented was a situation where a researcher uses an old protocol that calls for 1µl of proteinase K. While the protocol calls for that volume, she might actually require more or less of her enzyme, depending on enzyme activity. In the end, the article highlights important information defining enzyme activity, specific activity and other ways to evaluate proteinase K.
How Is Proteinase K Involved With Cell Lysis
To answer this question, the first thing to consider is what proteinase K is. Its a broad-spectrum protease capable of digesting a wide range of native proteins .
When it comes to cell lysis, particularly for downstream DNA isolation and purification, proteinase K can be part of the lysis step by digesting surface proteins. Further into the procedure, when it comes time to resuspend and lyse the nuclei in a buffer containing proteinase K, the proteinase K will help digest proteins that would otherwise degrade the sample.
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How To Use Recombinant Proteinase K For Your Nucleic Acid Extractions
Significant native Proteinase K shortages attributed to the COVID-19 pandemic have had far reaching effects across the life science research field . At this time, it is unknown how long the native product supply chain will remain inaccessible to manufacturers and researchers alike. Due to the sudden shortage, IBI Scientific now manufactures a quality, equitable product for life science researchers critical work– recombinant Proteinase K.
With IBI Scientifics recombinant Proteinase K, research world-wide proceeds unencumbered with results identical to that of native Pro-K. Not all recombinant Pro-K products are created equally, however. Keep reading to find out everything you need to know while using recombinant Proteinase K for your research.
What is recombinant Proteinase K and what can I use it for?
IBI Scientifics recombinant Proteinase K powder, originally isolated from the fungus Engyodontium album, is a serine protease with broad substrate specificity and relatively high proteolytic activity. It preferentially cleaves ester and peptide bonds adjacent to the C-termini of hydrophobic, aliphatic, or;aromatic amino acids. Proteinase K is used to digest proteins during RNA or DNA extraction and to inactivate nucleases that could degrade DNA or RNA. It also removes endotoxins and promotes cell lysis.
How does the enzymatic activity and stability of recombinant compare to native Pro-K?
What is important to know about how Pro-K has been expressed?
Dna Isolation From Tails
- Each tail should be in a clean eppendorf tube.
- Add 500 µl of tail lysis buffer containing;proteinase K to each tube.
- Incubate tail samples in 50-60°C water bath overnight.
- Add 250 µl saturated NaCl to each tube.
- Shake tubes vigorously and incubate tubes on ice for 10 minutes.
- Spin tubes on low speed at 4°C for 10 minutes.
- Remove supernatant and place into a clean eppendorf.
- Add 650 µl isopropanol and invert to mix. Incubate tubes at room temperature for 15 minutes.
- Recover DNA by centrifuging, max speed, 10 minutes at room temp.
- Place tubes inverted on bench and allow to air dry 5 minutes.
- Add 200 µl of TE pH 7.5 or sterile water to each tube. Incubate in 50-60°C water bath for 10 minutes. Re-suspend pellet by pipetting up and down several times.
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Do You Have Any Questions
Proteinase K is a proteolytic enzyme. The enzyme uses Ca2+ ions for structural stability. Due to the high total activity of the enzyme, lysis can be carried out in the presence of EDTA. Proteinase K is therefore excellently useful to digest contaminating proteins and improve the extraction of nucleic acids from various biological samples.
Proteinase K Antigen Retrieval Protocol
Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemically detection of certain proteins. The Proteinase K based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.
Solutions and Reagents:
- Proteinase K Solution TE Buffer :Tris Base – 6.10 gEDTA – 0.37 g
Triton X-100 – 5 ml
Mix to dissolve. Adjust pH 8.0 using concentrated HCl . Store at room temperature.
- Proteinase K Stock Solution Proteinase K – 0.008 g TE Buffer, pH8.0 – 10 mlGlycerol – 10 ml
Add Proteinase K to TE buffer until dissolved. Then add glycerol and mix well. Aliquot and store at 20°C for 2-3 years.
- Working Solution Proteinase K Stock Solution – 1 mlTE Buffer, pH8.0 – 19 ml
Mix well. This solution is stable for 6 months at 4°C.
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Does Edta Inactivate Proteinase K
This question also seems to come up quite a bit when discussing proteinase K. Chelators such as EDTA or EGTA dont have a direct effect on proteinase K enzyme activity.
Often, the reason for using EDTA with proteinase K during DNA or RNA purification is for the removal of calcium . But because calcium is related to proteinase K stability, the addition of EDTA can impact the calcium and therefore reduce proteinase K activity to some extent.
Role Of Proteinase K Digestion In Dna Extraction
Of course, it digests the protein, but how?
The major portion of the cell is made up of the protein. The nuclear membrane, enzymes, ligands, receptors, chaperons, antibodies and other receptor-specific legends are made up the only protein.;
Also, the cell membrane and nuclear membrane is made up of the composite protein such as phospholipid, glycoprotein and sphingomyelin. These are the several molecules which are made up of the proteins along with some lipids, carbohydrates and other molecules.
The proteinase K digest the protein portion of these molecules. Further, it digests the protein associated with DNA. The protein is made up of the long chain of amino acids. Depending upon their nature of interaction with water, it is categorised into the hydrophilic and hydrophobic amino acids.
The proteinase K digests mainly the hydrophobic amino acids . The cofactor, calcium ion provides stability to the enzyme, however, it cannot increase the activity of the reaction.
Highest proteinase K activity is reported at the temperature 60ºC ranging from 37ºC to 70ºC. The temperature vs enzymatic activity graph is shown below.
Furthermore, the stability of the enzyme reported highest at the pH 8.0 . However, the enzyme is active at pH 4.0 to up to 10.0. the graph of pH vs the activity of the enzyme is shown below,
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Proteinase K Dna Extraction Protocol
There are many variations in proteinase K DNA extraction protocol. However, the variation depends on the requirement of the researcher and the type of tissue.
I am describing the protocol for DNA extraction from the blood.
- Take 2 ml of the blood sample and add 10 to 20L of TE buffer to the sample. Mix well.
- Centrifuge the sample at 2500 rpm for 20 minutes
- Discard the supernatant and add 10 to 15L of TE buffer to the pallet and mix it gently.
- Centrifuge the sample at 2500 rpm for 15 minutes. Discard the supernatant and add 20L of proteinase K solution and 2 ml of DNA extraction buffer to the falcon tube.
- Incubate the sample at 56 to 60 for 1 to 2 hrs or until the pallet dissolve properly.
- Now add 1 to 2 ml of chilled isopropanol and a pinch of NaCl to the falcon tube and invert the tube for some time to obtain the precipitate.
- Centrifuge the tube at 8000 to 10,000 rpm to settle the pellet at the bottom.
- Discard the supernatant and add 1ml ethanol and centrifuge it at 10,000rpm for 1 to 2 minutes.
- Discard the ethanol and dry the pellet in the dryer now add TE buffer to the pellet and put it in a water bath for 2 to 3 hrs.
This protocol is for 2ml of the sample, you can design your own protocol for 1 ml or even 500L sample. calculate each chemical accordingly.;
The DNA extraction buffer composition varies from protocol to protocol as well. For the broad use include Tris, EDTA, NaCl, SDS or Triton X 100 or any other salt into the DNA extraction buffer.
What Exactly Is The Relationship Between Proteinase K And Calcium
Proteinase K binds to two Ca2+ ions which help maintain the stability of the enzyme, especially when its subjected to increasing temperatures. Calcium also protects proteinase K from autolysis. While calcium helps maintain proteinase K thermostability, it is not necessary for proteolytic activity.
According to Richard Tullis and Harvey Rubin, this relationship becomes more interesting when DNase I is involved. Proteinase K is known to inactivate DNases and RNases, but when DNase I is in the presence of Ca2+, it is protected from proteinase K . RNase, on the other hand, is inactivated whether or not it is in the presence of Ca2+. Their findings suggest a method for treating contaminated RNase free DNaseI or isolating highly polymerized RNA.
What Are The Guidelines For Using Pk
- 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. The enzyme is used for this method at a concentration of 1 mg/ml in a buffer containing 0.5M EDTA and 1% N-lauroylsarcosine . Incubate 24-48 hours at 37°C.
- 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 with proteinase K at a concentration of 1 mg/ml, for 12-18 hours at 50°C.
- 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% . Incubate for 30 minutes at 37°C. Total RNA can be isolated by passing the lysate through a needle fitted to a syringe prior treatment with the enzyme.
- 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 . Incubation is at 37°C for 30 minutes.
How Do You Inactivate Proteinase K
Inactivating proteinase K is perhaps one of the most common questions we see. And the answer is very simple. Heat is a widely used way of inactivating proteinase K. While the activity of proteinase K increases with temperature, and is optimized at about 65 C, heating proteinase K to 95 C for 10 minutes will inactivate it. Keep in mind, however, that heating proteinase K does not fully inactivate the enzyme. There will always be a small amount of activity remaining through this method.
Note- the actual inactivation temperature has been debated, ranging between 70 – 95 C. However, crowd sourced feedback and extensive research led us to settle on 95 C as the best temperature for inactivation.
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How Do You Determine If The Enzyme Is Working
To determine if the enzyme is working, you can do the following 2 steps:
Introduction To Dna Extraction:
DNA extraction is an important step in any genomic technique ranging from the polymerase chain reaction to microarray and from real-time PCR to next-generation sequencing.
For extracting DNA or isolating DNA from the cell we have to break cell membrane and nuclear membrane.
For breaking nuclear membrane as well as cell membrane different chemicals are utilised which are enlisted in the previous article: Different types of DNA extraction methods.
The first;DNA;extraction attempt had performed by;Friedrich Miescher;in 1869.
We can break the cell membrane and nuclear membrane of the cell by different methods such as mechanical method, chemical method and enzymatic method.
In this article, we will explain the enzymatic method in combination with the chemical method for extracting pure DNA.
Notably, obtaining High quality DNA is the prime importance of any; DNA extraction method. The DNA obtained from each DNA extraction method must be nearby ~1.80 260/280 absorbance ratio with more than 100g of concentration.
The detailed explanation of DNA extraction is discussed in The article: Different types of DNA extraction methods.; Read it first.
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Preparation Of Proteinase K Solution
The activity of enzyme decreases over a period of time so we have to store the proteinase K enzyme below 4ºC temperature. At the Lower temperature, the enzyme remains inactive.
The commercially available proteinase K enzyme comes in a powder form. It is extracted from the fungus. We have to prepare a stock solution as well as working proteinase K solution depending upon our requirements.
The proteinase K powder lyophilised either into the water or into the buffer.
If it is lyophilised into the buffer, we do not need to add any other extra ingredients such as buffer or glycerol. Just add the desired amount of water to the vial.
If it is lyophilised only into the water we have to add all other ingredients which are required for the optimum activity of the enzyme. Other ingredients are 10mM Tris , 1mM EDTA , 3mM CaCl2 and 50% glycerol. We can use TE buffer directly .
For the DNA extraction, 200g/ml of proteinase K working solution is sufficient. Generally, the proteinase K provided in 20mg/ml concentrations by the manufacturer.
Lets do some calculations,
20mg = 20,000g
20,000g/ 1000L is our stock solution
The requirement for the working solution is 200g/ml
C1= 20,000g/ 1000L
V1 is a volume given is unknown
C2 is concentration required = 200g
V2 is;volume;required= 1000l
V1= 1000 * 200/ 20,000
When we add 10L of proteinase K to the 990L water, the working solution becomes 200g/ ml, we can use 1L directly.
Applications For Molecular Biology:
- Detection of Bovine Spongiform Encephalopathy proteins which are uniquely resistant to proteolytic degradation.
- Tissue digestion as an alternative sample preparation approach for quantitative analysis using liquid chromatographytandem mass spectrometry.
- Specifically modify cell surface proteins to analyze membrane structures for protein localization
- Generates protein fragments used in characterization of functional studies
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