Among the spectrum of RNA modifications, N4-acetylcytidine (ac4C), also referred to as RNA ac4C modification, is notable for its distinctive acetylation role. This modification is facilitated by the enzyme NAT10 and is consistently present in tRNA and rRNA. Notably, ac4C exhibits a dynamic distribution in mRNA, which is modulated by developmental stages and changes in NAT10 expression levels.

Recent scientific progress has started to address the challenges associated with mRNA acetylation, highlighting its natural variations in cancer, developmental processes, and immune responses. These variations are fundamental to protein synthesis and cellular metabolism. The ac4C modification strengthens the stability of RNA molecules, thereby prolonging their cellular lifespan and enhancing their functional capacity. This improvement ensures the accurate and efficient decoding of genetic information into proteins. Furthermore, this modification influences the RNA structure, which could potentially alter its interactions with ribosomes and the translation machinery. Such alterations have a substantial impact on the efficiency and accuracy of protein synthesis, which are crucial for the proper functioning of cells and their ability to adapt to environmental changes.

The RayBio^® ac4C ELISA Kit is a competitive enzyme immunoassay developed for rapid detection and quantitation of ac4C in RNA, serum, plasma and cell medium samples collected from various species. The quantity of ac4C is determined by a known ac4C standard curve. This kit has a high sensitivity of approximately 0.707 ng/mL. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown samples.

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1-liter graduated cylinders
• Absorbent paper
• Distilled or deionized water
• GraphPad Prism or SigmaPlot software (or other software which can perform four-parameter logistic regression models)
• Tubes to prepare standard or sample dilutions
• Orbital shaker
• Aluminum foil
• Plastic wrap

1. Prepare all reagents, samples and standards as instructed.
2. Add 50 μL standards or samples to appropriate well. Incubate for 10 minutes at room temperature.
3. Add 50 μL anti-ac4C to appropriate well. Incubate 1 hour at room temperature. And wash 5 times.
4. Add 100 μL prepared HRP-Secondary antibody solution. Incubate 1 hour at room temperature. And wash 5 times.
5. Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 2-10 minutes at room temperature.
6. Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

The entire kit may be stored at -20°C to -80°C for up to 1 year from the date of shipment. For extended storage, it is recommended to store it at -80°C. Avoid repeated freeze-thaw cycles.