Agrisera - Model 5-fC -5-Formylcytosine

• Storage: Store at -20°C; make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tubes.
• Tested applications: DIP (DNA immunoprecipitation),  ELISA (ELISA)
• Related products: collection of antibodies to epigenetics
• Additional information: This serum contains 0.05 % sodium azide.

• Recommended dilution: 3 µl (DIP), 1: 10 000 (ELISA)
• Confirmed reactivity: human
• Predicted reactivity: mouse, broad species range
• Not reactive in: No confirmed exceptions from predicted reactivity known at the moment
• Additional information: To be added when available
• Selected references: To be added when available, antibody released in February 2016.

DIP: HEK293 cells were transfected with a reporter gene and hydroxymethylated in vitro with either a pCAG expression vector containing the TET2 catalytic domain (TET2cd) or a negative control pCAG vector. DIP assays were performed on 4 μg of sheared and denatured DNA using 3 μl of the polyclonal antibody against 5-fC in a total of 500 μl IP buffer. QPCR was performed with primers specific for the reporter gene. Figure 1 shows the recovery, expressed as a % of input (mean +standard deviation of 3 different experiments).

ELISA: to determine the titer of the antibody, an ELISA was performed using a serial dilution of the polyclonal antibody directed against 5-fC. The plates were coated with the immunogen. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be >1:100 000.