5-hydroxymethylcytosine (5-hmC), also known as the "sixth base" of DNA, is generated by the oxidation of 5-methylcytosine (5-mC) through the action of the ten-eleven translocation (TET) family of enzymes. 5-hmC is thought to be the DNA demethylation pathway, thereby playing a crucial part in gene regulation, chromatin structure and accessibility, recruiting or repelling transcription factors, and ultimately modulating gene expression. The presence of 5-hmC is particularly abundant in neuronal cells, suggesting a significant role in brain development and function. In addition, levels of 5-hmC undergo highly dynamic changes during development, differentiation and cancer. 5-hmC plays a critical role in developmental processes and dysregulation of 5-hmC has been associated with several human diseases.

The RayBio^® 5-hmC (5-Hydroxymethylcytosine) Dot Blot Assay Kit is a reliable, simple, and rapid method to quantify the level of 5-hmC in genomic DNA isolated from tissue, cells, urine, serum, plasma and other nucleic samples collected from humans, rats, mice, and other species.

• Highly sensitive and specific
• Requires minimal sample preparation
• No specialty equipment required
• Suitable for high-throughput screening
• Compatible with various sample types and species
• ≥ 48 samples could be detected by 1 kit

• RNase-free tubes
• Heat blocker
• 10 cm plastic petri dish
• UV cross-linker
• Chemiluminescent imaging system

1. Sample preparation: Depending on the type of sample (RNA, plasma, or serum), different methods of extraction, dilution, and denaturation are required.
2. Sample loading and crosslinking: The sample is spotted onto a nitrocellulose membrane, air dried, and crosslinked with UV light twice.
3. Methylene blue staining: One line of the spotted sample replicates is cut off and stained with methylene blue buffer to serve as an internal control.
4. Blocking and antibody incubation: The membrane is blocked with blocking buffer and then incubated with corresponding antibody and HRP-conjugated secondary antibody.
5. Detection and imaging: The membrane is treated with a mixture of detection buffers C and D, which catalyzes a color development reaction. The chemiluminescent signal is captured by an imaging system.

The entire kit may be stored at -20°C to -80°C for up to 12 months from the date of shipment. For extended storage, it is recommended to store it at -80°C. Avoid repeated freeze-thaw cycles.