The Epigenase™ APOBEC3 Cytidine Deaminase Activity/Inhibition Assay Kit is a complete set of optimized buffers and reagents for measuring the activity/inhibition of total APOBC3 enzymes using cell extracts or purified APOBEC3 isoforms (A3A, A3B, A3C, A3D, A3F, and A3H) from human and other mammals, in a variety of forms including, but not limited to, cultured cells and fresh/frozen tissues. The kit has the following advantages and features:
- Colorimetric assay with easy-to-follow steps that can be finished within 4 hours.
- Directly measures total APOBEC3 activity via a straightforward detection of APOBEC3-converted end products.
- Innovative kit composition enables background signals to be extremely low, allowing the assay to be simple, accurate, reliable, and consistent.
- Both cell/tissue extracts and purified APOBEC3 enzymes can be used for detecting APOBEC3 inhibitor effects in vivo and in vitro.
- Novel assay principle allows for high sensitivity with a detection limit as low as 10 ng of purified APOBEC3 enzymes.
- C-to-U converted standard is included for quantifying the specific activity of APOBEC3s.
- Strip microplate format for manual or high throughput analysis.
The APOBEC3 cytosine deaminases (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3) are a DNA/RNA editing family, which includes 7 subtypes: APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, and APOBEC3H. APOBEC3s are found exclusively in mammals and can catalyze the removal of an amino group from the cytosine (C) base to form uridine (U) in single-strand DNA or RNA. The genetic alterations by APOBEC3s will change transcription and mRNA processing and actively participate in various biological processes. APOBEC3s are part of the innate immune system against various DNA or RNA viruses to protect the integrity of cells. Through promoting the deamination of cytosine to uracil on ssDNA/RNA, APOBEC3s generate mutations in virus genomes to inhibit virus replication. For example, a C-to-U mutation caused by APOBEC3s in the SARS-CoV-2 genomes was reported to be the dominant mutation (>50%). While APOBEC3 confers its intrinsic host resistance to infection to various viruses, including the SARS-CoV-2 virus, it may also cause potential evolutionary consequences for viruses. APOBEC3s contribute significantly to DNA mutagenesis in cancer. As a major driver of cancer evolution, APOBEC3 proteins are one of the most predominant causes of genomic mutations detected in a variety of tumors. APOBEC3 mutation signatures can be seen in more than 50% of cancers, promoting tumor diversity that furthers disease progression and resistance to therapies.
Principle & Procedure
In this assay, a substrate is pre-coated onto microplate wells. Active APOBEC3s bind to the substrate and convert cytosine to uracil products. The APOBEC3-converted products can be recognized with a specific probe. The ratio or amount of uracil products, which is proportional to enzyme activity, can then be colorimetrically measured by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The activity of the APOBEC3 enzyme is, in turn, proportional to the optical density intensity measured.
The amount of cell extracts for each assay can be 2 µg to 30 µg with an optimal range of 15-20 µg. The amount of purified enzymes can be 10 ng to 1 µg, depending on the type, purity, and catalytic activity of the enzymes.
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