Enzymes and Inhibitors

<h2>What Are Nucleases?</h2>
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Nucleases are enzymes that cleave phosphodiester bonds between nucleotide subunits in nucleic acids. Deoxyribonucleases (DNases) act on DNA; ribonucleases (RNases) act on RNA; and some nucleases (S1 Nuclease, Mung Bean Nuclease) act on both single-stranded DNA and RNA. Nucleases are further divided into endonucleases, which cleave internal phosphodiester bonds, and exonucleases, which progressively remove nucleotides from the 5' or 3' terminus of a strand. Major research nucleases include DNase I (non-specific endonuclease for both ss- and ds-DNA), RNase A (cleaves single-stranded RNA at pyrimidine residues), RNase H (degrades the RNA strand of RNA: DNA hybrids), Exonuclease III (removes nucleotides from 3'-recessed or blunt ends of duplex DNA), S1 Nuclease (degrades single-stranded DNA or RNA), and RNase R (degrades linear RNA but not circular RNA). Match the nuclease to the substrate type (single-stranded vs. double-stranded, DNA vs. RNA), the directionality required (endo vs. exo), and cofactor compatibility with your reaction buffer.
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<h2 dir="ltr">What you will find:</h2>
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PureRec RNase A (Recombinant): A ribonuclease free of animal-derived components, designed for the swift elimination of RNA from DNA samples, ensuring no threat of viral contamination.
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DNase I Set (RNase-free): A highly effective endonuclease formulated for complete DNA digestion in delicate RNA applications while preserving the integrity of the transcript.
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PureRec Duplex-Specific Nuclease (DSN): A unique enzyme designed to equalize high-abundance transcripts, greatly enhancing the identification of rare variants in cDNA libraries.
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RNase R: A distinctive 3'-to-5' exoribonuclease that breaks down linear RNA but preserves circular RNA forms, crucial for the identification of circRNA.
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RNaseOFF Inhibitor: A strong protein-based barrier that counteracts a wide range of RNases, maintaining the delicate transcriptional environment of your samples.
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<h2>How to Choose Nucleases</h2>
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DNase selection
DNase I (bovine pancreatic, recombinant) non-specifically cleaves both single- and double-stranded DNA in the presence of Mg2+ (random cleavage) or Mn2+ (double-strand cuts at approximately the same position on both strands). Use RNase-free DNase I for RNA preparations to remove genomic DNA contamination. Use Plasmid-Safe ATP-Dependent DNase to selectively degrade linear double-stranded DNA while preserving supercoiled and relaxed circular plasmid DNA. Baseline-ZERO DNase is optimized for the complete removal of trace DNA without damaging RNA.
RNase selection
RNase A cleaves single-stranded RNA at pyrimidine (C and U) residues to produce 3'-pyrimidine nucleotides; it is used to remove RNA from DNA preps, to perform RNase protection assays, and to analyze RNA secondary structure. RNase H degrades the RNA strand of RNA: DNA duplexes and is used after first-strand cDNA synthesis to remove the mRNA template. RNase R (3'-&gt;5' exoribonuclease) degrades all linear RNA without affecting circular RNA, and is used in circular RNA enrichment workflows and to characterize circular RNA splice junctions. Terminator 5'-Phosphate-Dependent Exonuclease (Terminator) digests linear RNA with a 5'-monophosphate, enriching for capped mRNA or circular RNA.
Exonuclease selection
Exonuclease III (Exo III) removes nucleotides from the 3' terminus of duplex DNA and generates single-stranded regions used in unidirectional nested deletions, mapping regulatory elements, and generating single-stranded templates for sequencing. Exonuclease I (Exo I) digests single-stranded DNA in the 3'-&gt;5' direction and is used after PCR to remove unincorporated primers before Sanger sequencing. Lambda Exonuclease removes nucleotides from 5'-phosphorylated duplex DNA in the 5'-&gt;3' direction and is used in isothermal amplification and strand displacement workflows.
Cofactor and inhibitor sensitivity
DNase I requires Mg2+ or Ca2+ for activity and is completely inhibited by EDTA at a 5 mM concentration. RNase A is active across a wide pH range (optimum 7.0-7.5) and is exceptionally stable; inactivation requires proteinase K digestion, phenol-chloroform extraction, or RNase inhibitor protein. RNase H requires Mg2+; RNase R requires Mg2+ and is most active between 37-50 degrees C.
Purity and application grade
For RNA-focused applications, all nucleases used on DNA substrates must be certified RNase-free. For DNA-focused work, DNase-free grades of RNases are required. Confirm lot-specific QC contamination data from the manufacturer before use in sensitive workflows.
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<h2>Specifications Context</h2>
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DNase I activity is expressed in Kunitz units: 1 unit increases A260 by 0.001 per minute per mL during DNA hydrolysis at pH 5.0. RNase A activity is expressed as the amount of enzyme hydrolyzing 50% of an RNA substrate under defined conditions; it remains active at a wide range of temperatures and is notoriously difficult to inactivate (requires boiling in SDS, proteinase K digestion, or phenol-chloroform extraction). RNase R from E. coli degrades linear RNA processively in the 3'-&gt;5' direction and is used at 2-10 U per microgram of total RNA to enrich for circular RNA species. RNase R treatment before library construction is the standard method for circular RNA sequencing (circRNA-seq) workflows on both short-read and long-read platforms. For protecting RNA from RNase contamination during nuclease-adjacent workflows, see&nbsp;<a href="https://mbpinc.net/reagents/molecular-biology/enzymes-and-inhibitors">RNase inhibitors</a>&nbsp;in the parent Enzymes and Inhibitors category.
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Nucleases

DataSheet

MSDS

DNase I

Unit Quantity

Concentration

Note

Caution

Shipping Condition

MSDS - DNase I (RNase-Free)

DNase I (RNAse-Free)

<h2>What Are Restriction Enzymes?</h2>
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Restriction enzymes are endonucleases originally isolated from bacteria, where they function as a primitive immune defense against foreign DNA (such as bacteriophages) by cleaving at specific recognition sequences. Over 3,500 Type II restriction enzymes have been characterized, recognizing palindromic or asymmetric sequences of 4-8 base pairs and generating either 5' overhangs (sticky ends), 3' overhangs, or blunt ends. The systematic naming convention reflects enzyme origin: EcoRI comes from Escherichia coli strain R; HindIII from Haemophilus influenzae strain Rd. Restriction enzymes are grouped into Types I, II, III, and IV based on cofactor requirements, cleavage site position, and structural complexity; Type II enzymes are almost exclusively used in research applications due to their defined cleavage positions. Choose restriction enzymes based on the recognition sequence present in your target, the end type required (sticky vs. blunt), and whether Golden Gate Assembly or conventional cloning is planned.
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<h2 dir="ltr">What you will find:</h2>
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Enterokinase Cleavage Enzyme: A specific mammalian-produced protease for precise affinity tag removal from recombinant fusion proteins.
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DpnI: A specialized enzyme targeting methylated GATC sequences, ideal for template digestion in site-directed mutagenesis.
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Tn5 Transposase for Tagmentation: A high-efficiency enzyme for simultaneous DNA fragmentation and adapter ligation in NGS library construction.
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</ul>
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<h2>How to Choose Restriction Enzymes</h2>
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Type IIP vs. Type IIS
Type IIP enzymes (EcoRI, HindIII, BamHI, NcoI, NheI) recognize palindromic sequences and cut within or immediately adjacent to the site, making them the standard choice for conventional restriction cloning using compatible cohesive ends. Type IIS enzymes (BsaI, BsmBI/Esp3I, SapI) cut at a fixed distance outside their recognition sequence, generating custom 4-nt overhangs that can be designed to create any desired sequence at the assembly junction -- the basis of Golden Gate Assembly, which allows ordered multi-fragment cloning in a single tube.
Recognition sequence frequency
4-cutter enzymes (MboI, AluI, TaqI) cut on average every 256 bp and are used for genome-wide restriction mapping or chromatin fragmentation. 6-cutters (EcoRI, HindIII) cut every ~4,000 bp on average and are standard for plasmid cloning. 8-cutters (NotI, PacI, AscI) cut rarely (every ~65,000 bp) and are used for mapping of large genomic regions or release of large inserts from BAC or YAC vectors.
Star activity sensitivity
Restriction enzymes can exhibit relaxed sequence specificity (star activity) at suboptimal conditions: excess glycerol (&gt;5%), non-standard buffers, extended digestion times, or non-standard divalent cations. High-fidelity (HF) enzyme variants reduce star activity by more than 1000-fold and are recommended for long digestion times or when high specificity is critical for downstream cloning.
Isoschizomers and compatible ends
Isoschizomers recognize the same sequence as a reference enzyme; neoschizomers recognize the same sequence but cut at a different position. BamHI (GGATCC, 5' overhang G) is compatible with BglII (AGATCT) overhangs and with MboI and Sau3AI overhangs -- useful for directional cloning strategies without re-engineering sites. Check isoschizomer tables before designing multi-enzyme cloning strategies.
Buffer compatibility
Most suppliers have rationalized their enzyme buffers into 2-4 universal buffer systems (e.g., CutSmart, Buffer 3.1, FastDigest buffers) that support the majority of enzymes, enabling double digestion without buffer exchange. Verify buffer compatibility before combining two restriction enzymes in a single tube.
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<h2>Specifications Context</h2>
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Restriction enzyme activity is defined as the amount of enzyme required to digest 1 microgram of lambda phage DNA (or substrate specified by the manufacturer) to completion in 1 hour at 37 degrees C in 50 microliters total volume. FastDigest enzyme formats complete digestion in 5-15 minutes at 37 degrees C, using a universal buffer. Heat inactivation is possible for most enzymes at 65-80 degrees C for 20 minutes, though some enzymes (NheI, SpeI, BstEII) are not heat-inactivatable and require column cleanup before downstream ligation. Over 3,500 Type II restriction enzymes are characterized in the REBASE database; commercially available catalogues cover approximately 200-300 common enzymes. For ligating restriction-digested fragments, see&nbsp;<a href="https://mbpinc.net/reagents/molecular-biology/enzymes-and-inhibitors/dna-modifying-enzymes">DNA-modifying enzymes</a> for T4 DNA Ligase and alkaline phosphatase.
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Restriction Enzymes

DpnI

Thermostability Results for Enterokinase Activity

Enterokinase Cleavage Enzyme (Mammalian Produced)

Application

Storage Condition

<h2>What Are RNA-Modifying Enzymes?</h2>
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RNA-modifying enzymes act on RNA substrates to add, remove, or chemically alter nucleotides and terminal structures. Key enzymes in this category include poly(A) polymerase (PAP), which adds untemplated adenosine residues to RNA 3' ends; T4 RNA Ligase 1, which joins single-stranded RNA; T4 RNA Ligase 2, which joins nicked duplex RNA; vaccinia virus capping enzyme (VCE), which adds a 7-methylguanosine cap to the 5' end of mRNA; 2'-O-methyltransferase, which modifies the cap to generate a Cap 1 structure; and RNA methyltransferases such as METTL3/METTL14 complex used in m6A epitranscriptomics research. These enzymes are foundational tools in the fast-growing field of mRNA-based therapeutics and diagnostics, as well as long-read sequencing library construction. Select RNA-modifying enzymes based on RNA substrate type (single-stranded, duplex, capped), required end structure, and downstream application (therapeutic vs. research grade).
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<h2 dir="ltr">What you will find:</h2>
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T7 RNA Polymerase: The rapid powerhouse of in vitro transcription, ideal for producing large quantities of RNA from DNA templates for probes or functional mRNA research.
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Poly(A) Polymerase (Yeast &amp; E. coli): The definitive stabilizing agent, attaching adenosine tails to the 3' terminus of transcripts to enhance their longevity and translation efficacy&mdash;a crucial element for precise library preparation.
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<h2>How to Choose RNA-Modifying Enzymes</h2>
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Poly(A) tailing vs. RNA ligation.
Poly(A) polymerase is used when a 3'-poly(A) tail is needed for mRNA stabilization, translation enhancement, or as a primer binding site for oligo(dT)-based cDNA synthesis. T4 RNA Ligase 1 (single-strand-specific) is used for adaptor ligation to RNA 3' ends in small RNA sequencing library preparation. T4 RNA Ligase 2 truncated version (paired with a splint oligonucleotide) gives higher efficiency adaptor ligation with less secondary structure bias.
mRNA capping
5'-cap structures protect mRNA from exonucleolytic degradation and are recognized by eIF4E for ribosome recruitment. Vaccinia Capping Enzyme adds a Cap 0 structure; co-treatment with 2'-O-methyltransferase (mRNA Cap 2'-O-Methyltransferase) converts Cap 0 to Cap 1, which reduces innate immune recognition -- critical for therapeutic mRNA applications. Co-transcriptional capping with CleanCap analogs avoids the separate capping step.
Epitranscriptomics
m6A RNA methyltransferases (METTL3, METTL14, WTAP complex) and demethylases (FTO, ALKBH5) are used to add or remove N6-methyladenosine modifications in cellular RNA for functional studies. These enzymes require S-adenosylmethionine (SAM) as a methyl donor and are supplied at a microgram scale for biochemical assays.
RNase inhibitor pairing
All RNA-modifying enzyme reactions require an RNase-free environment. Include RNase inhibitor protein at 0.5-2 U per microliter in reactions without heat-inactivation capability. Use DEPC-treated water and certified RNase-free tubes and tips for all RNA work.
Application scale
Research-grade RNA-modifying enzymes are available in standard unit sizes for individual experiments. For manufacturing-adjacent workflows (large-scale IVT for mRNA vaccines or CAR-T cell therapy applications), contact MBP for volume pricing and GMP-compatible sourcing options.
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<h2>Specifications Context</h2>
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Poly(A) polymerase activity is measured as the amount of enzyme incorporating 1 nmol of AMP into acid-precipitable RNA in 30 minutes at 37 degrees C. Typical poly(A) tail lengths of 100-250 nucleotides are generated in 10-20 minute reactions at 37 degrees C with 1 mM ATP and 2-5 U of enzyme per microgram of RNA. Vaccinia Capping Enzyme reaction efficiency exceeds 95% for IVT transcripts of 500-5000 nt when used at recommended ratios; verify by LC-MS or HPLC analysis for therapeutic applications. Co-transcriptional Cap 1 capping using modified trinucleotide cap analogs is increasingly adopted in mRNA manufacturing to eliminate separate enzymatic capping steps.&nbsp;
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RNA-modifying enzymes

Poly(A) Polymerase, E. coli

Components

Storage Buffer

Poly(A) Polymerase, Yeast

<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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Proteases

Protease Inhibitor Cocktail

Recommended Protocol

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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