Product Details
| Alternate Name | Proteinase K, Endopeptidase K, Tritirachium alkaline proteinase, Protease K, Standard Grade Proteinase K |
|---|---|
| Gene Symbol | PROK |
| Gene ID | 5621 |
| Accession # | P06873 |
| Source | Recombinant Pichia pastoris |
| Appearance | Liquid |
| Physical Form Description | Liquid |
| Molecular Weight | 28.9 kDa |
| Purity by | >90% |
| Reconstitution Instructions | Soluble in water ((1–2 mg/ml) |
| Handling | Centrifuge the vial prior to opening. |
| Storage Conditions | -20ºC |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
A serine protease that displays the ability to digest native proteins, thereby inactivating enzymes such as DNase and RNase without recourse to a denaturation process. It retains its activity in presence of SDS and urea. It is inactivated by diisopropyl fluorophosphates (DFP) and phenyl methane sulfonyl fluoride (PMSF). It is not inhibited by iodoacetic acid, the trypsin-specific inhibitor TLCK, the chymotrypsin-specific inhibitor TPCK, and p-chloromercuribenzoate.
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What is the role of Proteinase K in DNA extraction?
Proteinase K, a broad spectrum serine protease, is used in DNA extraction protocols to digest the contaminating proteins, especially DNases. The enzyme cleaves the peptide bonds in proteins adjacent to the carboxyl group of aliphatic and aromatic amino acid residues.
What is the working concentration of Proteinase K?
The working concentration of Proteinase K can be anywhere between 50 - 400 µg/ml. The concentration of Proteinase K added depends on the tissue that is digested. A working concentration of 200 µg/ml is plenty for most tissues. The end user needs to optimize the best working concentration of proteinase K for theie sample type.
How to prepare a solution of Proteinase K?
Proteinase K is prepared as a 20 mg/ml stock in sterile water or in an appropriate buffer of choice. For DNA extraction, 200 μg/ml of Proteinase K working solution is sufficient. The activity of the enzyme decreases over time and so it is critical to store both the powder and solution at -20°C. Divide the enzyme solution into aliquots to avoid repeated freeze/thaw cycles. Additionally, BioVision offers ready to use Proteinase K solution (BioVision Cat# 9211, 9251).
What is the optimal temperature for Proteinase K digestion?
Proteinase K is active over a wide range of temperatures and buffers. The enzyme is active at 37°C and the activity increases with temperature up to 60C. Beyond 60C heating, will result in maximum loss of the enzyme activity.
What is the optimal pH for Proteinase K activity?
Proteinase K is active over a wide range of pH between 7.5 - 12,with an optimal activity at pH 8.0.
What are some of the applications of Proteinase K
The main application of Proteinase K is its use in DNA and RNA extraction. In most nucleic acid extraction protocols, Proteinase K is added to the tissue or the lysate to ensure complete digestion of proteins, especially DNases and RNases during nucleic acid extraction. The ability to digest proteins also makes Proteinase K useful for the preparation of chromosomal DNA for pulsed-field gel electrophoresis, protein fingerprinting, protease foot printing, mitochondrial isolation, genomic DNA isolation, etc.
How long should the samples be incubated with Proteinase K?
The duration of digestion with Proteinase K depends on the type of sample used. A cleared lysate following Proteinase K digestion is usually a sign that digestion has proceeded well. If the lysate is not clear, then duration should be extended. It is important to evaluate the exact incubation time to avoid over-digestion of samples. We strongly recommend standardizing the incubation times for your sample type.
How do you inactivate Proteinase K?
The most common way to inactivate Proteinase K is by heating. Once digestion is completed at an optimal temperature, samples are heated to 95°C for 10 minutes to inactivate the enzyme. It can also be inactivated by serine protease inhibitors such as diisopropyl fluorophosphates (DFP) and Phenylmethylsulfonyl fluoride (PMSF).
What is the recommended storage conditions for Proteinase K? How long is the enzyme stable at the recommended temperature ?
Proteinase K is an enzyme and hence proper storage is critical. The lyophilized solid can be stored desiccated at -20 ˚C for up to 2 years. Once you reconstitute the solid, aliquot your stock solution and store at -20 ˚C for up to 1 year. Also, it is advised to thaw the Proteinase K solution on ice and to avoid multiple freeze thaw cycles to prolong enzyme activity.
What is the activity of Proteinase K in commonly used buffers?
Based on the available literature, different buffer compositions have different impact on the activity of Proteinase K.
• Buffer: 30 mM Tris HCl, pH 8.0
Relative activity (approx.): 100%
• Buffer: 50mM Tris-HCl, pH 8.0, 1 mM CaCl2, 3 mM DTT, 2.0 M Urea
Application: Denaturation of proteins
Relative activity (approx.): 70%
• Buffer: 100 mM Tris-HCl, pH 8.0, 100 mM EDTA, 250mM NaCl, 1% Sarkosyl
Application: Plant tissue DNA isolation
Relative activity (approx.): 120%
• Buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% SDS
Application: Bacterial DNA isolation
Relative activity (approx.): 100%
• Buffer: 10 mM Tris HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.5% SDS 50 mM Tris-HCl, pH 8.0
Relative activity (approx.): 100%
• Buffer: 50 mM EDTA, 5% Tween 20, 0.5% Triton-X 100, 800 mM GuHCl
Relative activity (approx.): 300%
• Buffer: 30 mM Tris HCl, pH 8.0
• Buffer: 50mM Tris-HCl, pH 8.0, 1 mM CaCl2, 3 mM DTT, 2.0 M Urea
• Buffer: 100 mM Tris-HCl, pH 8.0, 100 mM EDTA, 250mM NaCl, 1% Sarkosyl
• Buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% SDS
• Buffer: 10 mM Tris HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.5% SDS 50 mM Tris-HCl, pH 8.0
• Buffer: 50 mM EDTA, 5% Tween 20, 0.5% Triton-X 100, 800 mM GuHCl
What are the differences between Proteinase K catalogue# 9210, 9211, 9247, 9250, 9251?
What is the role of EDTA in Proteinase K digestion?
EDTA inactivates the Proteinase K enzyme by chelating the divalent metal ions.
What is the molecular weight of Proteinase K?
28.9 kDa.
Why does Proteinase K not digest itself?
Proteinase K is sensitive to autolysis and is stabilized by the addition of calcium ions. In the absence of calcium, autolysis is promoted. Autolysis of the enzyme is observed during sample preparation for SDS-gel electrophoresis and at low concentration (0.01 mg/ml) in aqueous solution. It does not occur at higher Proteinase K concentrations at or above 1.0 mg/ml.
How do you test DNase/RNase contamination in Proteinase K?
We test DNase contamination using BioVision Cat# K429 and RNase contamination using BioVision Cat# K934.
