After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Specificity of various modified cytosine antibodies were determined by dot blot. The same sequence of a 387-base pair DNA fragment was generated by PCR using exclusively unmodified cytosine, 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-carboxylcytosine (5-caC), or 5-formylcytosine (5-fC). The respective DNA fragments were blotted onto a nylon membrane, UV cross-linked, and probed with 5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb #36836, 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692, 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660, and 5-Formylcytosine (5-fC) (D5D4K) Rabbit mAb #74178. As shown, the 5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb shows specificity for 5-caC.
|5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb 28692||20 µl||
|5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb 51660||20 µl||
|5-Carboxylcytosine (5-caC) (D7S8U) Rabbit mAb 36836||20 µl||
|5-Formylcytosine (5-fC) (D5D4K) Rabbit mAb 74178||20 µl||
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
|Anti-mouse IgG, HRP-linked Antibody 7076||100 µl||
The DNA Cytosine Modification Antibody Sampler Kit provides an economical means of detecting the levels of cytosine modifications in DNA by dot blot using antibodies against 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine.
5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb recognizes endogenous levels of 5-methylcytosine. 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb recognizes endogenous levels of 5-hydroxymethylcytosine. 5-Formylcytosine (5-fC) (D5D4K) Rabbit mAb recognizes transfected levels of 5-formylcytosine. 5-Caroxylcytosine (5-caC) (D7S8U) Rabbit mAb recognizes transfected levels of 5-methylcytosine. These antibodies have been validated using ELISA and dot blot, and cross-reactivity was not observed with other marks. Many cells and tissues contain very low endogenous levels of 5-hmC, 5-fC, and 5-caC that may fall below the detection limits of these antibodies.
Monoclonal antibodies are produced by immunizing animals with 5-methylcytidine, 5-hydroxymethylcytidine, 5-formyl-2'-deoxycytosine, or 5-carboxylcytidine.
Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1 (3, 4). 5-methylcytosine was originally thought to be passively depleted during DNA replication. However, subsequent studies have shown that Ten-Eleven Translocation (TET) proteins TET1, TET2, and TET3 can catalyze the oxidation of methylated cytosine to 5-hydroxymethylcytosine (5-hmC) (5). Additionally, TET proteins can further oxidize 5-hmC to form 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), both of which are excised by thymine-DNA glycosylase (TDG), effectively linking cytosine oxidation to the base excision repair pathway and supporting active cytosine demethylation (6,7).
TET protein-mediated cytosine hydroxymethylation was initially demonstrated in mouse brain and embryonic stem cells (5, 8). Since then this modification has been discovered in many tissues, with the highest levels found in the brain (9). While 5-fC and 5-caC appear to be short-lived intermediate species, there is mounting evidence showing that 5-hmC is a distinct epigenetic mark with various unique functions (10,11). The modified base itself is stable in vivo and interacts with various readers, including MeCP2 (11,12). The global level of 5-hmC increases during brain development and 5-hmC is enriched at promoter regions and poised enhancers. Furthermore, there is an inverse correlation between levels of 5-hmC and histone H3K9 and H3K27 trimethylation, suggesting a role for 5-hmC in gene activation (12). Lower amounts of 5-hmC have been reported in various cancers, including myeloid leukemia and melanoma (13,14).
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