Name: Proteinase K Recombinant Protein
Brand: CUSABIO
SKU: CSB-AP361972TIQ

Product Details

Description	Proteinase K, originally isolated from the mold Tritirachium 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. CUSABIO's recombinant proteinase K is highly pure determined by SDS-PAGE and reached up to 95%. It is characterized by high purity, sterility, no bio-burden, and no presence of DNAse, RNAse, DNA, and RNA contaminants. Its enzyme activity was calculated >30U/mg using the absorbance A275 as the vertical axis and different concentrations of a tyrosine as the horizontal axis. The working concentration of proteinase K, usually determined by the temperature, PH, and whether the buffer used contains SDS and urea, is 20mg/mL. This recombinant proteinase K protein is in-stock now. Proteinase K has a wide range of applications. In nucleic acid extraction and isolation, proteinase K can be used to degrade Dnase/Rnase and DNA-bound histones and lyse cells or tissues. It also digests protein impurities especially endotoxin and separates target nucleic acids from proteins in biological samples, thus improving the purity and safety of nucleic acids and production efficiency. The nucleic acids isolated by proteinase K is often used for the amplification reaction. Denaturing agents such as SDS (1%) can increase the enzymatic activity of proteinase K. In in-situ hybridization, it removes the proteins around the target DNA to facilitate probe infiltration and improves detection sensitivity. Proteinase K is also used to analyze the membrane structure through the modification of proteins on the cell surface. The nucleic acid-based real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test is the primary tool to diagnose the SRAS-CoV-2 infection. Proteinase K is often used to remove unwanted proteins present in the nasal/pharyngeal swab samples and inactivate nucleases that could degrade DNA or RNA during isolation and purification procedures, thus increasing the yield of extracted RNA and improving the detection of viral RNA by RT-PCR.
Purity	Greater than 95% as determined by SDS-PAGE.
Activity	Using the absorbance A275 as the vertical axis and different concentrations of tyrosine as the horizontal axis, a standard curve was drawn, and the enzyme activity was calculated ＞30U/mg.
Target Names	PROK
Uniprot No.	P06873
Alternative Names	Endopeptidase K,Tritirachium alkaline proteinase
Species	Tritirachium album (Engyodontium album)
Mol. Weight	28.9 kDa
Form	Lyophilized powder Note: We will preferentially ship the format that we have in stock, however, if you have any special requirement for the format, please remark your requirement when placing the order, we will prepare according to your demand.
Buffer	If the delivery form is liquid, the default storage buffer is Tris/PBS-based buffer, 5%-50% glycerol. Note: If you have any special requirement for the glycerol content, please remark when you place the order. If the delivery form is lyophilized powder, the buffer before lyophilization is Tris/PBS-based buffer, 6% Trehalose, pH 8.0.
Reconstitution	We recommend that this vial be briefly centrifuged prior to opening to bring the contents to the bottom. Please reconstitute protein in deionized sterile water.We recommend to add 5-50% of glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C. Our default final concentration of glycerol is 50%. Customers could use it as reference.
Troubleshooting and FAQs	Protein FAQs
Storage Condition	Store at -20°C upon receipt, aliquoting is necessary for mutiple use. Avoid repeated freeze-thaw cycles.
Shelf Life	The shelf life of the lyophilized form is 24 months at -20°C/-80°C, 18 months at 4°C, and 12 months at room temperature.
Lead Time	Basically, we can dispatch the products out in 3-7 working days after receiving your orders. Delivery time may differ from different purchasing way or location, please kindly consult your local distributors for specific delivery time.
Notes	Repeated freezing and thawing is not recommended.
Datasheet & COA	Please contact us to get it.

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■ Q&A

What is the activity of Proteinase K in common buffers?

Based on the available literature, different buffer (pH = 8.0, 50 °C, 1.25 µg/mL protease K, 15 min incubation) compositions have different impact on the activity of Proteinase K.
Buffer 1: 30 mM Tris·Cl
Proteinase K activity 1: 100%
Buffer 2: 30 mM Tris·Cl; 30 mM EDTA; 5% Tween 20; 0.5% Triton X-100; 800 mM GuHCl
Proteinase K activity 2: 313%
Buffer 3: 36 mM Tris·Cl; 36 mM EDTA; 5% Tween 20; 0.36% Triton X-100; 735 mM GuHCl
Proteinase K activity 3: 301%
Buffer 4: 10 mM Tris·Cl; 25 mM EDTA; 100 mM NaCl; 0.5% SDS
Proteinase K activity 4: 128%
Buffer 5: 10 mM Tris·Cl; 100 mM EDTA; 20 mM NaCl; 1% Sarkosyl
Proteinase K activity 5: 74%
Buffer 6: 10 mM Tris·Cl; 50 mM KCl; 1.5 mM MgCl2; 0.45% Tween 20; 0.5% Triton X-100
Proteinase K activity 6: 106%
Buffer 7: 10 mM Tris·Cl; 100 mM EDTA; 0.5% SDS
Proteinase K activity 7: 120%
Buffer 8: 30 mM Tris·Cl; 10 mM EDTA; 1% SDS
Proteinase K activity 8: 203%

What is the recommended pH for Proteinase K to keep its enzyme activity?

Proteinase K is a proteolytic enzyme that is purified from the Tritirachium album. Proteinase K is stable over a wide pH range 4.0–12.5 with an optimum of pH 8.0.

What is the main applications of Proteinase K?

Proteinase K is a serine protease that is mainly used in DNA and RNA extraction. In most nucleic acid extraction protocols, Proteinase K is added to the tissue or the lysate to digest proteins and remove contamination (especially DNases and RNases) from nucleic acid preparations. Additionally, Proteinase K also is useful for the preparation of chromosomal DNA for next generation sequencing (ngs) and microarray technologies.

How to dissolve proteinase K powder? What should I do if the dissolution is not complete or the dissolution time is longer ?

Proteinase K powder can be dissolved in the solution with 20mM Tris-HCl, 200mM NaCl, 50% glycerol, pH8.4. In this recommended diluent formulation, CUSABIO proteinase K lyophilized powder can be fully dissolved.After dissolution, it can be stored for a year at 4 ℃. If the client can use it up within 15 days, it can be directly used in pure water with 50% glycerin.

What is the long-term storage temperature of proteinase K from CUSABIO?

The form of proteinase K from CUSABIO is powder. The powder can be stored for 18 months at 4℃ and for more than 2 years -20℃.

Whether the form of proteinase K is liquid or powder, will repeated freezing and thawing affect the activity?

We don't recommend repeated freeze-thaw, and all of protein products are not suitable for repeated freeze-thaw. Compared with liquid protease K, it has less effect on Proteinase K powder, but please avoid moisture absorption.

What is the recommended working concentration of Proteinase K from CUSABIO?

The recommended working concentration is 20mg/mL.

What is the optimal temperature for proteinase K activation? And how to inactivate proteinase K?

Generally, the activity of proteinase K increases with temperature. The optimal temperature for activity ranges between 50-65 ˚C. Once the temperature beyond 65 ˚C, as temperatures increase, you risk inactivating proteinase K. Heating proteinase K to 95 ˚C for 10 minutes will inactivate it. However, heating proteinase K does not fully inactivate the enzyme. It still has a small amount of activity remaining through this method.

Proteinase K digest protein and remove contamination, but it doesn’t digest itself. Why?

Actually, proteinase K is sensitive for autolysis. The enzyme is stabilized by the presence of calcium ions. Absence of calcium promotes autolysis of the enzyme and also reduces the half-life of the enzyme activity. However, proteinase K is not completely inactivated by autolysis. Some enzyme fragments continue to maintain their proteolytic activity, even after extensive autolysis.

How long do the samples need incubating with Proteinase K?

The time of samples incubating with proteinase K is depended on the extraction you desired.
Isolation of high molecular weight DNA: You can treat the chromosomal DNA embedded in agarose 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 (v/v). Incubate the sample with proteinase K for 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 with 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 for 12-18 hours at 50°C.
Isolation of RNA: After centrifuging the cell lysate and removing the supernate, cytoplasmic RNA can be isolated by adding 200 ug/ml proteinase K and SDS to 2% (w/v) and Incubating for 30 minutes at 37°C.
Inactivation of RNases, DNases and enzymes in reactions: The enzyme should be used at a ratio of approximately 1:50 (proteinase K: enzyme). Incubation is at 37°C for 30 minutes.

Does EDTA inactivate proteinase K?

Commonly, in the process of DNA or RNA purification, using EDTA with proteinase K is to remove calcium. But since calcium is related to the stability of proteinase K, the addition of EDTA can impact the calcium, thus reducing proteinase K activity to certain extent.

How does Proteinase K works in DNA extraction?

The role of Proteinase K in DNA extraction is to digest many contaminating proteins present. Moreover, it also degrades nucleases presented in DNA extraction and protects the nucleic acids from nuclease attack.

Can proteinase K degrade DNA?

Instead of degrading the DNA, protease K digests the proteins surrounding the DNA, allowing it to be isolated intact.

What does proteinase K do to the cell?

Proteinase K can lyse or break down the cell membrane and release the intercellular materials including DNA and RNA.

How do you use proteinase K?

Dissolve lyophilized proteinase K in deionized sterile water. We recommend to add 10 mM Tris HCl, pH 7.5, 20 mM calcium chloride, and 50% glycerol, and then aliquot for long-term storage at -20/-80°C.

Why do we denature proteinase K?

Because proteinase K may degrade the useful proteins in the downstream applications of isolated nucleic acids.

How do you make proteinase K buffer?

The best buffer for proteinase K will be different in various applications. Proteinase K is active over a wide pH range (4-12.5) and relatively high temperature (optimum temperature 50-65 ˚C). Generally, proteinase K is stable and keeps highly active in buffers that contain denaturing reagents such as urea, sodium dodecyl sulfate (SDS), and guanidinium salts.

Why do we add proteinase K to our DNA extractions?

Proteinase K is used to lyse cells, disrupt tissue integrity, and degrade various proteins to make the pure DNA is fully released during DNA extraction. It also inactivates DNase present in the sample, ensuring DNA whole and intact. The wide temperature range and relatively strong pH tolerance of proteinase K also contribute to its extensive availability in nucleic acid extraction.

Target Data

Function	Hydrolyzes keratin at aromatic and hydrophobic residues.
Protein Families	Peptidase S8 family

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Code	CSB-AP361972TIQ
Size
Image	SDS-PAGE (Tris-Glycine gel) Discontinuous SDS-PAGE (reduced) with 5% enrichment gel and 15% separation gel. According to the left SDS-PAGE image, the purity of Proteinase K is 95%+ and the molecular weight is 28.9 kDa. Detect Nucleic acid residue by agarose gel electrophores Detect DNase residue by agarose gel electrophores Detect RNase residue by agarose gel electrophores Using the absorbance A275 as the vertical axis and different concentrations of tyrosine as the horizontal axis, a standard curve was drawn, and the enzyme activity was calculated ＞30U/mg. Unit definition:One unit is defined as the amount of enzyme that catalyzes the formation of 1 umol of tyrosine per minute at pH 7.5 at 37°C The Enzyme Activity Stability of Proteinase K The Proteinase K powder was stored at 4℃. And its enzyme activity was assayed every month over the past year. The left chart shows how its enzyme activity changes in a year. It turns out the enzyme activity is maintained more than 95%, which indicates that proteinase K is very stable. The Proteinase K activity comparison of 3 vendors and CUSABIO CUSABIO performed a Proteinase K activity comparison test. As the left chart shows, the activity of CUSABIO Proteinase K is better than that of the 3 vendors under the same testing conditions.
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Proteinase K Recombinant Protein

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