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

Cat. No.: PAGMN-300k (for 300KU)

Description

Protein AG-MNase, also known as Protein AG-Micrococcal Nuclease (pAG-MNase), is a fusion protein that combines Protein A/G and Micrococcal Nuclease (MNase). It possesses both the antibody binding activity of Protein A/G and the DNA cleavage activity of MNase. It is commonly used in the study of protein-DNA interactions, such as Chromatin Immunocleavage (ChIC) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN).

We also offer Protein A-MNase (pA-MNase) and Protein G-MNase (pG-MNase) simultaneously.

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), usually at the Fc region of the immunoglobulin. However, some data indicate that Protein A can also bind to the Fab region of the human VH3 family, and Protein G may occasionally bind to the Fab region as well. Additionally, their binding capacities differ for different subclasses of immunoglobulins. Overall, both Protein A and Protein G have wide applicability for most antibodies.

Micrococcal Nuclease (MNase), also known as S7 Nuclease, is a nuclease derived from Staphylococcus aureus. It can degrade various forms of DNA or RNA, such as single-stranded, double-stranded, linear, circular, etc., and generate 3'-phosphate ends of mononucleotides and oligonucleotides under pH 7.0-10.0 and Ca2+ conditions. MNase exhibits higher cutting efficiency for single-stranded nucleic acids than double-stranded nucleic acids. MNase shows approximately 30 times higher cutting efficiency on the 5' side of adenine (A), thymine (T), or uracil (U) compared to 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, among other applications. Furthermore, MNase only 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. The MNase provided by BioCloud is expressed and purified using their proprietary PerfectProtein™ technology platform. The recombinant protein obtained from expression and purification is identical to the natural Staphylococcus aureus MNase in terms of amino acid sequence and biochemical properties. It does not have any additional tags or amino acids.

This product is a 1:1 mixture of fusion proteins combining Protein A or G with MNase, and it possesses dual activities of Protein A/G antibody binding and MNase DNA cleavage.

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℃, 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 pAG-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