Description
Proteinase K is a highly reactive nonspecific serine protease that belongs to the subtilisin family of proteins. It cleaves at the carboxylic acid side of aliphatic, aromatic, or hydrophobic amino acids. Proteinase K is capable of inactivating RNases and DNases and is used in the isolation or preparation of high molecular weight nucleic acids. Proteinase K is also useful for helping to characterize enzymes, due to its cleavage specificity. This enzyme was designated proteinase K because of its ability to hydrolyze keratin. Proteinase K is stable in a wide variety of detergents and buffer salts and at various temperatures and pH. The isoelectric point of proteinase K is 8.9.
Proteinase K is typically used at a concentration of 50-100 μg/ml. It is active with or without the presence of SDS, urea, EDTA or various metal ions, but the activity of proteinase K can be increased by adding the denaturing agents and the structure of proteinase K can be stabilized by addition of Ca 2+. Proteinase K is inactivated by heating to 95°C for 10 minutes or using an inhibitor such as PMSF, AEBSF or DFP.
GoldBio's Proteinase K undergoes two sterile filtration steps during the separation and purification processes. The final product is further analyzed by plate culture to ensure the removal of all living organisms was 100% effective.
Unit Definition: One unit is defined as the amount of enzyme that will liberate 1.0 μmol tyrosine (Folin-positive amino acid) from casein per minute at 37°C, pH 7.5.
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Functional Highlights and Mechanism
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Proteinase K is a serine protease: its catalytic triad (Ser, His, Asp) enables a nucleophilic attack on peptide bonds, leading to broad substrate specificity.
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It cleaves after aliphatic, aromatic or hydrophobic residues, enabling digestion of diverse proteins—including nucleases, histones, membrane proteins and protein-bound nucleic acids.
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It retains activity in the presence of detergents (e.g., SDS), urea, EDTA and several buffer systems—making it suitable for challenging sample preparations.
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The enzyme is thermostable relative to many proteases: its activity increases with temperature (up to ~65 °C) which facilitates enhanced digestion of proteins in complex or aggregated states.
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It also inactivates RNases and DNases by degrading them, thereby protecting the integrity of nucleic acid targets during isolation workflows.
Recommended Applications and Usage Notes
Recommended Applications
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Digestion of unwanted proteins for preparations of genomic DNA, cytoplasmic RNA or high-molecular-weight nucleic acids.
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Inactivation and removal of RNases/DNases during extraction protocols (e.g., from tissue, blood, plants, microbial cells) to maximize yield and purity.
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Protease footprinting or characterization of protein interactions, due to its broad cleavage specificity.
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Sample preparation for prion research (TSE transmissible spongiform encephalopathies), mitochondrial isolation, and other specialized workflows requiring robust protease activity.
Usage Tips and Considerations
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Dissolve the lyophilized powder in an appropriate buffer (e.g., 50 mM Tris-HCl, pH 8.0 plus 3 mM CaCl₂) to prepare a stock solution (e.g., 20 mg/mL) and filter-sterilize if required.
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Aliquot stocks to avoid repeated freeze-thaw cycles and store at -20 °C. Lyophilized powder can remain stable for up to ~2 years if stored desiccated at -20 °C.
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For optimum activity, consider the temperature: incubate at ~50-65 °C when performing digestion of tight/aggregated proteins. Many standard protocols still operate at 37 °C, but higher temperature speeds the process.
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Add Ca²⁺ (e.g., 3 mM CaCl₂) to stabilize the enzyme and prevent autolysis; detergents and denaturants (SDS, urea) can enhance activity by exposing protein substrates.
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When proceeding with downstream nucleic acid work, ensure the protease is properly inactivated (e.g., by heating to 95 °C for 10 minutes or using inhibitors such as PMSF or AEBSF) to avoid unintended proteolysis.
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Choose between powder and solution formats based on convenience vs cost: powder allows more flexibility and lower cost per unit volume; solution offers convenience and less prep time.
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Always consult the lot-specific Certificate of Analysis provided by GoldBio to confirm activity units, purity, and absence of nucleases (especially important for ultra-clean nucleic acid work).
Common Research Applications
(Click each for more information)
Genomic DNA Isolation and Purification
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Purpose: To eliminate protein contaminants during DNA extraction and ensure high-purity nucleic acid yields.
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How It Works: Proteinase K hydrolyzes peptide bonds adjacent to aliphatic and aromatic amino acids, degrading histones, nucleases, and other proteins that interfere with DNA isolation. Its compatibility with detergents like SDS and chelators like EDTA allows for efficient cell lysis and protein digestion.
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Applications: Extraction of high-quality genomic DNA from tissues, whole blood, bacteria, and cultured cells for PCR, sequencing, and cloning.
Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press.
RNA Isolation for RT-PCR and Transcriptomics
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Purpose: To protect RNA from degradation by eliminating ribonucleases during RNA purification workflows.
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How It Works: Proteinase K is added during or after lysis to degrade residual RNases. It remains active under denaturing conditions and is commonly used with guanidinium thiocyanate-containing reagents to ensure RNA integrity.
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Applications: Isolation of total RNA from animal and plant tissues, cell cultures, and clinical samples for transcriptome analysis, RT-qPCR, and RNA-seq.
Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry, 162(1), 156–159.
Protein Digestion in Histological and In Situ Hybridization Preparations
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Purpose: To enhance probe and antibody penetration by enzymatically softening fixed tissue sections.
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How It Works: In formalin-fixed, paraffin-embedded or cryopreserved tissues, Proteinase K is applied prior to hybridization or staining. It digests cross-linked proteins and structural matrix components that obstruct access to intracellular targets, while preserving nucleic acid integrity. The protease is typically used under tightly controlled conditions to balance tissue preservation with signal enhancement.
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Applications: Fluorescence and chromogenic in situ hybridization (FISH, CISH), immunohistochemistry (IHC), and TUNEL assays to localize gene expression, detect transcripts, or assess apoptosis in intact tissue sections.
Levsky, J. M., & Singer, R. H. (2003). Fluorescence in situ hybridization: past, present and future. Journal of Cell Science, 116(14), 2833–2838.
Organelle Isolation and Analysis
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Purpose: To remove cytosolic or peripheral membrane proteins during subcellular fractionation, leaving internal components intact.
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How It Works: Low concentrations of Proteinase K are used to digest surface proteins or contaminants after organelle isolation (e.g., mitochondria, nuclei), enabling studies of internal components without full degradation.
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Applications: Verification of membrane integrity in mitochondrial or nuclear fractions, proteomic profiling of organelles, and quality control of subcellular preparations.
Frezza, C., Cipolat, S., & Scorrano, L. (2007). Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nature Protocols, 2(2), 287–295.
Inactivation of DNases and RNases in Reagent Preparation
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Purpose: To sterilize laboratory reagents and solutions by enzymatically degrading contaminating nucleases that can degrade target nucleic acids.
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How It Works: Proteinase K is used to digest residual nucleases in solutions such as water or buffer components, followed by heat inactivation or organic extraction to remove the protease itself.
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Applications: Preparation of DEPC-treated water, nuclease-free buffers, and plastics used in PCR, qPCR, and other nucleic acid assays.
Wilson, I. G. (1997). Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology, 63(10), 3741–3751.
Application Summary
- Digestion of unwanted proteins
- Removal of endotoxins bound to cationic proteins such as lysozyme and RNaseA
- Removal of nucleases for in situ hybridization
- Prion research with respect to TSE (transmissible spongiform encephalopathies)
- Protease footprinting Mitochontrial isolation
- Isolation of genomic DNA
- Isolation of cytoplasmic RNA
- Isolation of highly native DNA or RNA
Storage/Handling
Store at -20°C.
Reconstitute in 50mM Tris-HCl (pH 8.0), 3mM CaCl2.
Proteinase K Quick Answers:
How do you inactivate proteinase K?
Inactivate proteinase K by heating it to 95˚C for 10 minutes. You can also permanently inactivate proteinase K by using protease inhibitors such as PMSF and AEBSF (Pefabloc®).
What is the best temperature for proteinase K activation?
The best temperature for proteinase K activity is between 50-65˚C. Higher temperatures help with protein unfolding, which makes it easier for proteinase K to break down proteins.
How do you store proteinase K?
Aliquot proteinase K stock solution and store at -20˚C for up to 1 year.
Store lyophilized proteinase K powder desiccated at -20˚C for up to 2 years.
How do you dissolve proteinase K?
Proteinase K is water soluble and it can also be dissolved in Tris or PBS. Dissolving proteinase K with PBS is a little challenging, which may be due to pH. It's best to add proteinase K a little bit at a time while mixing into solution.
What is the optimal pH when working with proteinase K?
The optimal pH when working with proteinase K is between 7.5 - 12.0
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