| Product Description | Aladdin's Protein A-MNase (pA-MNase) is a fusion protein of Protein A and Micrococcal Nuclease (MNase), with both Protein A antibody binding and MNase endonuclease activities. It is commonly used in ChIC (Chromatin Immunocleavage) and CUT&RUN (Cleavage Under Targets and Release Using Nuclease) assays for protein-DNA interaction studies.aladdin also provides Protein G-MNase (pG-MNase) and Protein AG-MNase (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 in Streptococcal bacteria (type C or G). Protein A and Protein G are functionally similar and bind specifically to the mammalian immunoglobulins (Ig) in the Fc region usually. However, it has been shown that Protein A also binds to the Fab region of the human VH3 family, and protein G as well. Although Protein A and Protein G differ in their ability to bind immunoglobulins in some subclasses, they are applicable to most antibodies. For detailed information about protein A and protein G, please refer to the manual of aladdin' s Protein A+G Agarose or UltraBio™ Protein A+G Magnetic Beads .Micrococcal Nuclease (MNase), also known as S7 Nuclease, is an endonuclease derived from Staphylococcus aureus. It can degrade single-stranded, double-stranded, linear and circular DNA or RNA in the presence of Ca2+ at pH 7.0-10.0, producing single nucleotides or oligonucleotides with 3' phosphate groups. MNase cleaves single-stranded nucleic acids more efficiently than double-stranded nucleic acids. The cleavage efficiency of MNase on the 5' side of adenine (A), thymine (T) or uracil (U) is about 30 times higher than cleaving guanine (G) or cytosine (C). It cleaves more efficiently on AT or AU-rich regions. Therefore, as a "relatively "non-specific" nucleic acid endonucleases, the MNase is commonly used to remove nucleic acids from cell lysates. In addition, MNase only cleaves DNA on the linker region between nucleosomes, and thus more often used for chromatin fragmentation in ChIP assays. This product is expressed and purified with the PerfectProtein™ Platform developed by aladdin. The amino acid sequence of MNase and its biochemical properties are exactly the same as the wild type version from Staphylococcus aureus.CUT&RUN (Cleavage under Targets and Release Using Nucleases) is a fast, effective and reliable technical method for detecting protein-DNA interactions in cells. Cells are first immobilized on Concanavalin A magnetic beads (ConA magnetic beads) and then permeabilized with a detergent such as Digitonin, followed by the addition of specific primary antibody and pA-MNase or pG-MNase. The primary antibody recruits pA-MNase or pG-MNase to the target protein on chromatin, and the MNase is activated by Ca2+ at appropriate concentrations and cleaves the DNA on both sides of the target protein to release the cleaved genomic DNA which is then enriched and analyzed by qPCR or next generation sequencing (NGS). Compared with traditional chromatin immunoprecipitation (ChIP) assay, CUT&RUN has the advantages of time saving, less sample required, lower background for NGS sequencing, and good reproducibility, and therefore has now been extensively used in gene transcriptional regulation and epigenetic studies [1-3].Please refer to Figure 1 for the performance of this product.Figure 1. Degradation of Lambda DNA by Aladdin's Protein A-MNase (pA-MNase) . In 20μl reactions containing 50mM Tris pH8.0, 5mM CaCl2, and 1μg Lambda DNA, different amounts of pA-MNase were added as indicated. After 15min incubation at 37℃, place the reactions immediately on ice and add 1μl of 0.5M EDTA to stop reactions. The reaction products were examined by agarose gel electrophoresis after adding the DNA Loading Buffer . This figure is for reference only, which may vary due to different experimental conditions.Definition of enzyme activity unit
Enzyme storage buffer： 5mM Tris (pH7.4), 50mM NaCl, 1mM EDTA, 50% glycerol.
Precautions： This product contains 50% glycerol and will not freeze when stored at -20℃. Storage at -80℃ must be avoided. Freeze-thaws may reduce its enzyme activity.This product is viscous. Please be careful when aspirating to ensure exact amount is taken. Mix well after adding to samples and avoid the formation of air bubbles.Ca2+ is a necessary cofactor for MNase activity. Metal ion chelators such as EDTA and EGTA that can bind calcium should be avoided in the reaction.The salt ion concentration in the reaction mix must be less than 100 mM. Too high concentration of salts will affect the enzyme activity of MNase.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： pA-MNase is mainly used for CUT&RUN assays. For the detailed procedure, please refer to the manual of 's CUT&RUN Assay Kit .
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