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Cat. No.: MN-320k (for 320KU)

Cat. No.: MN-1600k (for 1600KU)

Description

Micrococcal Nuclease (MNase), also known as Micrococcal Endonuclease or S7 Nuclease, is a nucleic acid endonuclease derived from Staphylococcus aureus. It has a CAS number of 9013-53-0 and a molecular weight of approximately 18.6 kDa. MNase can degrade various forms of DNA or RNA, including single-stranded, double-stranded, linear, circular, etc., under pH 7.0-10.0 and Ca2+ conditions, generating 3'-phosphate ends of mononucleotides and oligonucleotides. MNase exhibits higher cutting efficiency for single-stranded nucleic acids than double-stranded nucleic acids. Its cutting efficiency on the 5' side of adenine (A), thymine (T), or uracil (U) is approximately 30 times higher than guanine (G) or cytosine (C), making it effective in cleaving regions rich in AT or AU content. Therefore, MNase is considered a "relatively" non-specific nuclease and is commonly used for the removal of nucleic acids from cell lysates. Additionally, MNase selectively cleaves the DNA in the linker region of nucleosomes, while the DNA on the nucleosomes is protected by histones and not susceptible to MNase cleavage. Therefore, MNase is also commonly used for chromatin fragmentation in chromatin immunoprecipitation experiments.

The principle of Chromatin Immunoprecipitation (ChIP) is to cross-link or leave non-cross-linked protein-DNA complexes in live cells. The chromatin is randomly fragmented into small fragments using ultrasound or MNase treatment. Then, specific antibodies are used to immunoprecipitate DNA fragments bound to the target protein. Various downstream detection techniques such as quantitative PCR, PCR, Southern blotting, gene chips, and high-throughput sequencing are employed to analyze the sequences of the immunoprecipitated DNA fragments. ChIP is typically used to investigate whether genomic DNA-binding proteins such as transcription factors or histones are present in the same complex as specific genomic DNA sequences such as promoters or enhancers. It is crucial for studying gene transcription regulation and epigenetics.

Chromatin immunoprecipitation can be divided into cross-linked ChIP (X-ChIP) and native ChIP (N-ChIP) based on the different treatments of chromatin. Cross-linked ChIP involves cross-linking chromatin with formaldehyde, followed by ultrasound or MNase treatment to obtain DNA fragments of 200-1000 bp. It is suitable for studying proteins with weaker DNA or histone affinities. Cross-linking helps to maintain these proteins in their proper positions and prevents them from dissociating from their DNA binding sites. Native ChIP, on the other hand, does not require cross-linking. Untreated chromatin is usually treated with MNase to fragment the chromatin into segments containing 1-5 nucleosomes (approximately 200 bp per nucleosome). This method is suitable for studying specific DNA binding sites of histone modifications. The advantages and disadvantages of these two chromatin immunoprecipitation methods are summarized in the following table, and the selection can be based on the experimental purpose.

The recombinant protein obtained through expression and purification has a purity higher than 98% (SDS-PAGE). It has an amino acid sequence that is identical to the natural Staphylococcus aureus MNase. There are no additional tags or amino acids present, and it exhibits the same biochemical characteristics as the natural Micrococcal Nuclease. MNase is advantageous for chromatin immunoprecipitation experiments due to its good integrity and gentle nature. The cleavage of MNase on the DNA linker between nucleosomes preserves the integrity of the nucleosomes. The enzymatic digestion of chromatin by MNase is milder, eliminating the variability in ultrasonic power and the negative effects of chromatin emulsification during the ultrasonic process. This approach avoids protein denaturation and degradation caused by ultrasound, which can disrupt antigenic epitopes and compromise effective antibody binding.

Activity Definition

One Agarose Gel Unit is defined as the amount of enzyme required to digest 1µg of Lambda DNA in 15 minutes at 37℃, to the extent that the accumulation of low molecular DNA fragments is <400 base pairs as determined by agarose gel electrophoresis. Another Unit is Kunitz Unit. One Kunitz Unit is defined as the amount of enzyme required to release acid soluble oligonucleotides that produce an absorbance increase of O.D. 1.0 at 260nm in 30 minutes at 37℃. 1000 Agarose Gel Units is approximately equal to 100 Kunitz Units.

Enzyme Storage Buffer

5mM Tris (pH 7.4), 50mM NaCl, 1mM EDTA, 50% Glycerol. BSA (100X) is provided in this product for dilution of MNase.

Reaction Buffer (10X)

500mM Tris (pH 8.0), 50mM CaCl2.

Inactivation or Inhibition

The enzyme can be inactivated by heating at 65°C for 10 minutes or by adding an appropriate amount of EDTA or EGTA.

For chromatin immunoprecipitation experiments, if using 0.5-1μl of MNase (2000 gel units/μl) for 4×106 cells, the small packaging (D7201S) of this product can be used for 160-320 reactions, and the medium packaging (D7201M) can be used for 800-1600 reactions.

Precautions

• This product contains 50% glycerol and will not freeze at -20°C. Avoid storing at -80°C as freeze-thaw cycles may reduce the enzyme activity.
• The product has a viscous consistency. When pipetting, ensure accurate sampling, and after adding the sample, mix thoroughly by gentle tapping to avoid bubble formation.
• Ca2+ is a critical cofactor for MNase's catalytic activity. The reaction buffer containing 1-5mM Ca2+ is necessary for optimal enzyme activity. Metal chelators such as EDTA or EGTA in the reaction solution can affect enzyme activity.
• The salt ion concentration in the reaction solution should be below 100mM. Excessive salt concentration can affect MNase's enzyme activity.
• If the sample does not contain proteins, add BSA at a final concentration of 1X to the reaction buffer.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment. It should not be used for food or drugs and should not be stored in a regular household.
• For your safety and health, please wear appropriate lab attire and disposable gloves when handling.

Storage

The minimum shelf life is 1 year at -20°C.