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Cat. No.: PAMN-60k (for 60KU)

Cat. No.: PAMN-300k (for 300KU)

Description

Protein A-Micrococcal Nuclease, also known as Protein A-MNase or pA-MNase, is a fusion product of Protein A and Micrococcal Nuclease (MNase). It combines the antibody binding activity of Protein A with the DNA endonuclease activity of MNase. It is commonly used in ChIC (Chromatin Immunocleavage) and CUT&RUN (Cleavage Under Targets and Release Using Nuclease) assays for studying protein-DNA interactions.

We also offer Protein G-Micrococcal Nuclease (pG-MNase) and Protein A/G-Micrococcal Nuclease (pAG-MNase).

Protein A is a cell wall surface protein found in Staphylococcus aureus with a molecular weight of 42 kDa. Protein G is an immunoglobulin-binding protein expressed by C-type or G-type streptococcal bacteria. Protein A and Protein G have similar functions and can specifically bind to mammalian immunoglobulins (Ig). The binding sites are usually located in the Fc region of the immunoglobulin, but there is evidence that Protein A can also bind to the Fab region of human VH3 family. Protein G, on the other hand, sometimes also binds to the Fab region. Additionally, their binding capabilities vary for different subclasses of immunoglobulins. Overall, Protein A and Protein G are widely applicable for most antibodies. 

Micrococcal Nuclease (MNase), also known as S7 Nuclease, is a DNA endonuclease derived from Staphylococcus aureus. It can degrade various forms of DNA or RNA, including single-stranded, double-stranded, linear, and circular, under pH 7.0-10.0 and Ca2+ conditions, generating 3'-phosphate ends of mononucleotides and oligonucleotides. MNase has higher cutting efficiency for single-stranded nucleic acids than double-stranded nucleic acids. The cutting efficiency at the 5' side of adenine (A), thymine (T), or uracil (U) is approximately 30 times higher than that at guanine (G) or cytosine (C), making it suitable for digestion of AT- or AU-rich regions. Therefore, MNase is considered a "relatively" nonspecific DNA endonuclease and is commonly used for removing nucleic acids from cell lysates. Moreover, MNase only cleaves DNA in the linker regions between nucleosomes, while DNA on nucleosomes is protected by histones and is not susceptible to MNase digestion. The MNase product provided by BioVision is expressed and purified using the PerfectProtein™ technology platform, and the amino acid sequence is identical to the natural Staphylococcus aureus MNase. It does not contain any additional tags or amino acids and has the same biochemical properties as native Micrococcal Nuclease.

This product is a fusion product of Protein A and MNase, possessing the dual activities of antibody binding and DNA endonuclease of MNase.

CUT&RUN stands for Cleavage Under Targets and Release Using Nuclease, which is a rapid, efficient, and reliable technique for studying protein-DNA interactions in cells. The principle of CUT&RUN involves immobilizing cells on ConA magnetic beads, permeabilizing the cell membrane with detergent such as Digitonin, and adding specific primary antibodies and Protein A-MNase or Protein G-MNase. The primary antibodies recruit pA-MNase or pG-MNase to the target protein on chromatin. Then, MNase is activated by an appropriate concentration of Ca2+, resulting in cleavage of the DNA on both sides of the target protein and release of the cleaved genomic DNA. The subsequently purified and enriched cleaved genomic DNA fragments can be detected and quantified by qPCR or used for next-generation sequencing (NGS) analysis. Compared to traditional chromatin immunoprecipitation (ChIP), CUT&RUN offers advantages such as time efficiency, minimal sample requirement, low NGS sequencing background, and good experimental reproducibility. It is widely used in the study of gene transcription regulation and epigenetics.

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°C, 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 Solution

5mM Tris (pH7.4), 50mM NaCl, 1mM EDTA, 50% Glycerol.

When used in CUT&RUN experiments, this product is calculated based on using 1.5μl of pA-MNase (2000 gel units/μl) per reaction. The small package of this product is sufficient for 20 reactions, and the medium package is sufficient for 100 reactions.

Precautions

• This product contains 50% glycerol and will not freeze when stored at -20°C. Avoid storing at -80°C as it may cause freezing, and repeated freezing and thawing may affect the enzyme activity.
• This product is viscous, so ensure accurate sample volume when aspirating. After adding the sample, thoroughly mix by pipetting up and down to avoid bubble formation.
• Ca2+ is a crucial cofactor for MNase activity. The reaction buffer containing 1-5mM Ca2+ is necessary for optimal MNase 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. High salt concentrations can affect MNase activity.
• This product is intended for scientific research purposes by professionals only. It should not be used for clinical diagnosis or treatment, food or drug purposes, and should not be stored in residential areas.
• For your safety and health, please wear laboratory attire and disposable gloves when handling this product.

Storage

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

Related: 

• Protein A-MNase
• Protein G-MNase
• Protein AG-MNase