Proteases

<h2>What Are Proteases?</h2>
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Proteases (also called proteinases or peptidases) catalyze the hydrolysis of peptide bonds in proteins and polypeptides. They are classified by their catalytic mechanism into serine proteases (Proteinase K, trypsin, chymotrypsin, subtilisin), cysteine proteases (papain, calpain), aspartic proteases (pepsin), metalloproteases (thermolysin, carboxypeptidase A), and threonine proteases. In research applications, proteases serve roles that span nucleic acid purification, proteomics sample preparation, cell culture maintenance, recombinant protein processing, and structural biology. Trypsin cleaves at the carboxyl side of lysine and arginine residues and generates peptides in the 500-3,000 Da mass range ideal for LC-MS/MS peptide identification. Select a protease based on substrate specificity (broad-spectrum vs. site-specific), compatibility with your lysis or reaction buffer, and the need for downstream inactivation.
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<h2 dir="ltr">What you will find:</h2>
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<a href="https://mbpinc.net/reagents/molecular-biology/enzymes-and-inhibitors/proteases/proteinase-k" title="Proteinase K:">Proteinase K:</a> A robust serine protease (available in lyophilized or liquid formats) designed for rapid protein digestion and the inactivation of nucleases during sensitive nucleic acid isolation.
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Aprotinin, A high-purity competitive inhibitor of serine proteases, is essential for preventing unwanted proteolytic activity during delicate protein purification and extraction steps.
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Protease Inhibitor Cocktail, A pre-formulated, multi-component blend engineered to provide immediate, broad-range protection against a variety of endogenous proteases in cellular lysates.
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TEV Protease, A specialized endopeptidase with ultra-fine specificity, is utilized for the precise, site-directed cleavage of affinity tags from recombinant fusion proteins.
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<h2>How to Choose Proteases</h2>
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Nucleic acid purification
Proteinase K is the first choice for nucleic acid extraction because it is a broad-spectrum serine endopeptidase that digests nucleases (DNases, RNases), histones, and structural proteins in the presence of SDS, urea, EDTA, and most detergents. It is active from pH 4.0 to 12.5 with optimal activity at pH 8.0, and at temperatures from 20 to 60 degrees C. Inactivation at 95 degrees C for 5 minutes or by proteinase K inhibitors (PMSF) is required before downstream enzymatic steps.
Proteomics and mass spectrometry
Trypsin (sequencing grade) is the industry standard for bottom-up proteomics: it generates predictable lysine/arginine-terminated peptides of 6-25 amino acids, well-suited for LC-MS/MS analysis on Orbitrap and Q-TOF platforms. Chymotrypsin (cleaves after Phe, Tyr, Trp, Leu) and Glu-C/V8 protease (cleaves after Glu in phosphate buffer, or Glu+Asp in bicarbonate) are used for orthogonal digestions to improve protein sequence coverage. Lys-C protease (cleaves after Lys only) is stable in 8 M urea and is used for denatured or hard-to-digest proteins.
Cell culture dissociation
Trypsin at 0.25% (w/v) with 0.53 mM EDTA is the standard reagent for detaching adherent mammalian cells from culture surfaces. Recombinant trypsin formulations avoid the lot variability and animal-derived pathogen concerns of porcine pancreatic trypsin. Collagenase (types I, II, III, IV) is used for tissue dissociation, particularly for primary cell isolation from solid tissues, including liver, pancreas, breast, and adipose.
Recombinant protein processing
Site-specific proteases are used to remove N-terminal affinity tags (His6, GST, MBP) from purified recombinant proteins without leaving extra residues. Thrombin cleaves after Arg in the sequence LVPR/GS; Factor Xa cleaves after Arg in IE/DGR; TEV protease cleaves between Gln and Ser in ENLYFQ/S. TEV is the most commonly used because of its stringent 7-amino-acid recognition sequence, which minimizes off-target cleavage.
Buffer compatibility
Confirm protease compatibility with your lysis buffer before use. Proteinase K retains activity in 1% SDS, 4 M urea, 1% Triton X-100, and 10 mM EDTA. Trypsin is inhibited by EDTA (chelates the Ca2+ that stabilizes trypsin structure) and by soybean trypsin inhibitor (SBTI). Chymotrypsin is inhibited by PMSF (an irreversible serine protease inhibitor). Factor Xa is inhibited by benzamidine.
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<h2>Specifications Context</h2>
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Trypsin activity for proteomics is expressed in TAME units (N-alpha-p-tosyl-L-arginine methyl ester esterase units); 1 TAME unit hydrolyzes 1 micromol of TAME per minute at 25 degrees C, pH 8.1. Sequencing-grade trypsin is treated with TPCK (tosylphenylalanyl chloromethyl ketone) to irreversibly inactivate contaminating chymotrypsin activity, which would otherwise generate non-tryptic peptides that complicate database searches. Proteinase K activity is expressed as mAnson units per mL or mg, where 1 mAnson unit releases 1 micromol of tyrosine equivalents per minute from hemoglobin at pH 7.5 and 35.5 degrees C. Single-pot, solid-phase-enhanced sample preparation (SP3) using magnetic bead-bound proteases is increasingly adopted in proteomics to reduce sample loss and processing time for low-input samples.&nbsp;
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MSDS

Protease Inhibitor Cocktail

Protease Inhibitor Cocktail G135 — ready-to-use liquid solution for broad protease inhibition during cell lysis. Available without EDTA (G135) or with EDTA (G135E). Recommended: 1 ml per 100 ml lysate at 10⁸ cells/ml. Store at −20 °C, stable 1 year.

Unit Quantity

Recommended Protocol

Storage Condition

Shipping condition

Protease Inhibition Cocktail with EDTA

<h2>What Is Proteinase K?</h2>
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Proteinase K is a subtilisin-related serine endopeptidase with a molecular weight of 28,930 daltons (28.9 kDa), first isolated from Tritirachium album Limber in 1974, and named for its ability to digest keratin ('K') as its original substrate. The enzyme cleaves peptide bonds at the carboxylic side of aliphatic, aromatic, and basic amino acid residues, making it effective against nearly all protein substrates, including histones, chromatin-bound proteins, cell membrane proteins, and contaminating nucleases (DNases, RNases). Critically, Proteinase K retains full activity in the presence of 1% SDS, 4 M urea, 1% Triton X-100, 0.5% N-lauroylsarcosine, EDTA at 5 mM, and common reducing agents -- conditions under which most other proteases are inactivated. Use Proteinase K in any protocol requiring enzymatic removal of proteins from nucleic acid preparations, including genomic DNA, total RNA, plasmid, phage, and cfDNA extraction from tissues, blood, FFPE samples, bacteria, yeast, and environmental samples.
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<h2 dir="ltr">What you will find:</h2>
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Proteinase K (Lyophilized): A stable, high-purity powder ideal for prolonged storage and tailored buffer preparation.
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Proteinase K Solution: A prepared, stable liquid that reduces preparation time and minimizes pipetting mistakes.
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Proteinase K (G029): A specially refined grade designed for the swift inactivation of RNases and DNases in sensitive procedures.
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<h2>How to Choose Proteinase K</h2>
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Solution vs. lyophilized
Proteinase K is supplied as lyophilized powder (commonly at 30 mAnson units/mg or 20 mg/mL equivalent activity) or as a ready-to-use solution (typically 20 mg/mL in storage buffer). Lyophilized powder offers longer shelf life (up to 2 years at -20 degrees C), cost efficiency for high-volume users, and flexibility in buffer preparation. Solutions are convenient for low-volume use and eliminate weighing errors. Both formats deliver equivalent specific activity when the lot certificate is checked.
Molecular biology grade vs. standard
Molecular biology grade Proteinase K is certified free of detectable endonuclease, exonuclease, DNase, and RNase activities by stringent lot-release assays. Standard grades may carry trace nuclease activity that degrades DNA or RNA during digestion. For any application where nucleic acid integrity matters -- DNA sequencing, NGS library prep, RT-PCR, FISH, or southern blotting -- use only molecular biology grade or equivalent.
Recombinant vs. native
Recombinant Proteinase K (expressed in Pichia pastoris or E. coli) offers freedom from animal-derived material, greater lot-to-lot consistency, and suitability for regulated or GMP-adjacent workflows. Native Proteinase K (isolated from T. album culture) is standard for most research applications. Both must carry DNase/RNase-free certification for nucleic acid use.
Concentration and unit dose
Standard working concentrations are 50-200 microgram/mL for most DNA/RNA extraction protocols in lysis buffer. Higher concentrations of 1-5 mg/mL are used for FFPE tissue deparaffinization and protein digestion, and for prion inactivation studies (where Proteinase K at 10-50 microgram/mL is used to assess proteinase K-resistant PrP accumulation). Proteinase K retains activity for up to 24 months when stored at -20 degrees C in aliquots.
Temperature and pH optimization
Optimal activity is at 20-60 degrees C and pH 7.5-12.0; activity at pH 8.0 and 55 degrees C is used in most nucleic acid extraction protocols. Activity at 37 degrees C is approximately 80% of the 55-60 degrees C optimum. Calcium ions (1 mM Ca2+) stabilize the enzyme structure; EDTA removes calcium and reduces activity, though the enzyme retains sufficient function for nucleic acid prep protocols. Heat-inactivation above 95 degrees C for 5 minutes is complete and does not require chemical inhibitors.
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<h2>Specifications Context</h2>
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Proteinase K specific activity is commonly reported in Anson units (mAnson units/mg): one mAnson unit releases 1 micromol of tyrosine equivalents per minute from hemoglobin at pH 7.5 and 35.5 degrees C. Proteinase K is fully denatured at 95 degrees C in 5 minutes without requiring PMSF or other chemical inhibitors, making heat inactivation the simplest and most reliable option before downstream enzymatic steps such as PCR or reverse transcription. The enzyme is deactivated above 95 degrees C within the first cycle of PCR thermocycling, preventing interference with polymerase activity in protocols that include Proteinase K in the PCR reaction. Proteinase K is a universal reagent in automated nucleic acid extraction platforms, including MagNA Pure, KingFisher, and Hamilton NGS workstations; solution-format Proteinase K is specified in most automated platform kits.&nbsp;
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Proteinase K

DataSheet

G029 Proteinase K 20 mg/ml by ABM — serine protease for protein digestion and nuclease inactivation in DNA/RNA extraction. 1.0 ml; store at 2–8°C (short term) or −20°C up to 1 year (long term). For genomic DNA, RNA isolation, FFPE extraction, and ChIP.

Concentration

TEV Protease

E027 by Applied Biological Materials is an enhanced TEV Protease at 10 U/µl (100 µl; 1000 U total), supplied with 20X Reaction Buffer and 100 mM DTT. Cleaves ENLYFQ(G/S) with up to 99% efficiency in 1–2 hours at 30°C; active 4–30°C and pH 5.5–9.0. N-terminal 6X-His tag enables one-step Ni-IDA removal post-cleavage (use with Cat. No. G250). Shipped on Ice Pack; store at &minus;20°C.