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

Cat. No.: PGMN-300k (for 300KU)

Description

Protein G-Micrococcal Nuclease (pG-MNase) is a fusion protein consisting of Protein G and Micrococcal Nuclease (MNase). It combines the antibody binding activity of Protein G with the DNA endonuclease activity of MNase. It is commonly used in Chromatin Immunocleavage (ChIC) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) experiments for studying protein-DNA interactions.

We also offer Protein A-Micrococcal Nuclease (pA-MNase) and Protein AG-Micrococcal Nuclease (pAG-MNase).

Protein A is a cell wall surface protein found in Staphylococcus aureus with a molecular weight of 42kDa. Protein G is an immunoglobulin-binding protein expressed by C-type or G-type Streptococcal bacteria. Both Protein A and Protein G share similar functions and can selectively bind to mammalian immunoglobulins (Ig), typically at the Fc region of the antibodies. However, there is evidence that Protein A can also bind to the Fab region of the human VH3 antibody family, and Protein G may have some binding affinity to the Fab region as well. Additionally, their binding capacities may vary for different subclasses of immunoglobulins. Overall, Protein A and Protein G are widely applicable to most antibodies.

Micrococcal Nuclease (MNase), also known as S7 Nuclease, is an endonuclease derived from Staphylococcus aureus. It can degrade various forms of DNA or RNA, including single-stranded, double-stranded, linear, and circular structures, under conditions of pH 7.0-10.0 and in the presence of Ca2+. MNase produces 3'-phosphate ends of mononucleotides and oligonucleotides. It exhibits higher efficiency in cutting single-stranded DNA compared to double-stranded DNA. MNase shows approximately 30-fold higher cutting efficiency at the 5' side of adenine (A), thymine (T), or uracil (U) compared to guanine (G) or cytosine (C). It is particularly effective in cleaving regions rich in AT or AU content. Therefore, MNase is considered a "relatively" non-specific endonuclease and is commonly used for the removal of nucleic acids from cell lysates, among other applications. MNase only cleaves DNA in the linker regions between nucleosomes, while the DNA within nucleosomes remains protected by histones and is not susceptible to MNase digestion. The MNase provided in this product is expressed and purified using Bioyun Tian's proprietary PerfectProtein™ technology platform. The recombinant protein obtained exhibits the same biochemical properties as the native Staphylococcus aureus MNase, with no additional tags or amino acids.

This product is a fusion protein of Protein G and MNase, possessing both the antibody binding activity of Protein G and the endonuclease activity 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°C. 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 pG-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