Proteinase K

Proteinase K is a broad-spectrum, subtilisin-related serine protease derived from the fungus Parengyodontium album (Tritirachium album). The enzyme is active across a wide range of temperatures, pH values and denaturing conditions (including SDS and EDTA). Its high specific activity enables efficient digestion of proteins, including DNases and RNases, during nucleic acid preparation without compromising DNA or RNA integrity.  

The recombinant 28.9 kDa enzyme is expressed in Komagataella phaffii (Pichia pastoris) and extensively purified for high solubility, specific activity and low residual DNA content. QIAGEN Proteinase K is available in ready-to-use solution or lyophilized powder formats, including an Ultrapure option. 

Proteinase K - Solution can be provided in bulk and as a customized product with an extended shelf life of up to 3 years. We also offer Proteinase K - Powder as a lyophilized powder in a range of pack sizes to maximize long-term stability, maintain high enzymatic activity and permit ambient temperature shipping.  

Contact us for more information about custom and OEM options. 

QIAGEN Protease is an economical endopeptidase alternative to Proteinase K and is suitable for isolation of native DNA and RNA from a variety of biological samples, e.g., blood and cultured cells. 

High-quality sample preparation is an essential step before molecular analysis of target genetic material. Proteinase K plays an important role throughout the multiple stages of this procedure. To assure the highest purity of nucleic acids, the initial focus should be on the certified quality of Proteinase K. 

QIAGEN Proteinase K (RUO) in 2 mL and 10 mL pack sizes (cat. nos. 19131 and 19133) is dedicated for use in various QIAGEN kits (see list in the Principle section below). QIAGEN Proteinase K (cat. no. 19134) is available as a ready-to-use 10 mL solution for laboratory use and is included in QIAsymphony DSP Circulating DNA Kits (see list in the Principle section below). These enzymes are supplied at 20 mg/mL and offer high activity (>600 mAU/mL) in buffers commonly used in most DNA and RNA isolation procedures. The products are stable at room temperature for convenient workflow integration and are quality-guaranteed by QIAGEN.  

Proteinase K – Powder in lyophilized format offers high activity with exceptional lot-to-lot consistency (see figure “ High lot-to-lot consistency Proteinase K powder”) ensuring reproducibility for stable working conditions (see figure “ Proteinase K powder stability”). 

Proteinase K – Solution in a ready-to-use liquid format is stable at room temperature for convenient workflow integration (see figure “ High stability Proteinase K liquid”). An established manufacturing process ensures high lot-to-lot consistency of this product (see figure “ High lot-to-lot consistency Proteinase K liquid”).  

Proteinase K – Ultrapure in lyophilized format with enhanced purity and solubility is ideal for highly sensitive applications requiring minimal residual DNA.

* One mAU is the activity that releases folin-positive amino acids and peptides corresponding to 1 µmol tyrosine per minute.

† One unit of Proteinase K hydrolyzes urea-denatured hemoglobin producing the color equivalent of 1 μmol tyrosine per 1 minute at 37°C and pH 7.5 (Folin & Ciocalteu’s method), 1 U = 1 mAnsonU.

Proteinase K cleaves peptide bonds adjacent to the carboxyl group of aliphatic, aromatic and other hydrophobic amino acids. It is routinely used for the isolation of genomic, plasmid and high molecular weight DNA and RNA, as well as for the digestion of structural proteins and chromatins.

QIAGEN Proteinase K (RUO) (cat. nos. 19131 and 19133) is included in the following QIAGEN kits:

• QIAamp Circulating Nucleic Acid Kit
• QIAamp DNA Mini Kit
• QIAamp Fast DNA Stool Mini Kit
• DNeasy Blood & Tissue Kit
• QIAamp DNA Micro Kit
• QIAamp MinElute Media Kit
• QIAamp Media MDx Kit

QIAGEN Proteinase K (catalog no. 19134) is included in the following QIAGEN kits:

• QIAsymphony DSP Circulating DNA Kit
• QIAsymphony DSP Circulating DNA Maxi Kit

Stock solution preparation: Proteinase K – Powder and Proteinase K - Ultrapure

• 20 mg/mL solutions: Use nuclease-free water for immediate use in a sterile nuclease-free tube
• 20–50 mg/mL solutions for immediate use: 50 mM Tris-HCl, pH = 7.5–8.0, 1-5 mM Ca²⁺ (calcium chloride, calcium acetate)
• 20–50 mg/mL solutions for long-term storage: 10 mM Tris-HCl, pH = 7.5–8.0, 1-5 mM Ca²⁺ (calcium chloride, calcium acetate), 50% glycerol

Considerations for use

1. The enzyme is typically used at 50–200 μg/mL nucleic acid preparations at pH 7.5–8.5 and 37–55°C. Incubation times vary from 30 minutes to 18 hours.
2. Proteinase K cleaves proteins preferably behind hydrophobic amino acids. The smallest peptide to be hydrolyzed is a tetrapeptide. 
3. Working pH range is 4.0–12.0 with optimum activity at pH 7.5–8.5. Full activity is maintained over several hours over a pH range of 6.5–10.0.
4. Working temperature range is 20–65°C with optimum activity at 50–56°C.
5. The enzyme is stimulated by addition of denaturing agents 0.2–1% SDS or 1–4M urea. It exhibits prolonged stability due to the presence of Ca²⁺ (1–6 mM), which protects enzyme from autolysis and increases its thermal stability.
6. The enzyme is not inactivated by chelating agents (e.g. EDTA), chaotropic salts, detergents (e.g. 1% SDS, 1–4M urea), metal ions, thiol reagents or trypsin-specific inhibitors.
7. Proteinase K is usually denatured by subsequent phenol extractions. It can be also inactivated by heating above 65°C or using inhibitors such as PMSF or DIFP.

Proteinase K is highly effective at removing protein contamination due to its activity between 50–65°C in a wide pH range (7.5–12.0) combined with stability in the presence of denaturing reagents (SDS, urea) and chelating agents (EDTA).

Applications include:

• Removing protein contaminants in PCR, sequencing and NGS workflows.
• Cell lysis and tissue digestion in combination with SDS and detergents to isolate genomic DNA and cytoplasmic RNA from tissues and cultured cells.
• Rapid inactivation of DNases and RNases during RNA/DNA purification.
• Degrading proteins (e.g., histones) that bind to DNA in blood to improve DNA yield and purity for downstream analysis.
• Included in single-use cartridges for other automated molecular diagnostic devices that integrate sample preparation, amplification and detection.