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.

• 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

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Lyophilized Standard
• Capture Antibody
• Assay Diluent
• Biotinylated Peptide
• HRP-Streptavidin
• Positive Control
• TMB One-Step Substrate Reagent
• Stop Solution

• 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
• 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 100 µL Capture Antibody to each well. Incubate 1.5 hours at room temperature or overnight at 4°C.
3. Add 100 µL standard or sample to each well. Incubate 2.5 hours at room temperature or overnight at 4°C.
4. Add 100 µL prepared Streptavidin solution. Incubate 45 minutes at room temperature.
5. Add 100 µL TMB One-Step Substrate Reagent to each well. Incubate 30 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.