5-hmC Antibody - BSA Free

Dot Blot: 5-hmC Antibody [NBP2-59184] - To demonstrate the specificity of the antibody against 5-hmC, a Dot blot analysis was performed using hmC, mC and C controls. One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane (Amersham Hybond-N+). The antibody was used at a dilution of 1:1,000. Figure shows a high specificity of the antibody for the hydroxymethylated control.

Methylated DNA Immunoprecipitation: 5-hmC Antibody [NBP2-59184]

Methylated DNA Immunoprecipitation: 5-hmC Antibody [NBP2-59184] - hMeDIP (hydroxymethylated DNA IP) was performed using the antibody against 5-hydroxymethylcytosine. DNA from mouse ES cells was prepared and sonicated to obtain DNA fragments of 300-500 bp. One ug of sheared DNA was spiked with the unmethylated (C) methylated (mC), and hydroxymethylated (hmC) controls. hMeDIP was performed with 3.5 ug of the rabbit 5-hmC antibody and the IP'd DNA was analysed by qPCR using primers specific for the 3 different control sequences. Figure shows that the rabbit polyclonal antibody against 5-hmC is highly specific for the 5-hmC base modification (no IP with non-methylated or methylated C control fragments).

ELISA: 5-hmC Antibody [NBP2-59184] - To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against 5-hmC, crude serum and flow through, in antigen coated wells. The antigen used was the 5-hmC base coupled to BSA. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:2,800.