5-Methylcytosine antibody (mAb) (Clone 33D3) tested by Methyl-DNA Immunoprecipitation (MeDIP). Human DNA (500 ng) digested with Mse I was subjected to MeDIP and then analyzed by quantitative real-time PCR with primers specific for genes that are normally methylated (Xist, NYESO and SNRPN) or unmethylated (APC and p48). The MeDIP'd DNA (enriched DNA) was plotted.
5-Methylcytosine (5-mc) antibody (pAb) tested by Immunohistochemistry Punctate nuclear staining pattern is detected in Formalin-fixed, paraffin-embedded tissue sections from human substantia nigra (midbrain). Top Panel: 5-mC antibody at 1:1000 dilution. Bottom Panel: No primary antibody (2nd step antibody alone)
Next-Gen sequencing data generated using 5-Methylcytosine (5-mC) antibody (mAb) correlates well with CpG density. DNA was enriched from 1 ug of denatured, adaptor ligated human PC9 cell DNA using 2 ul of the 5-Methylcytosine (5-mC) antibody (mAb). MeDIP DNA was sequenced using the Illumina platform to generate 26 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows that the enriched regions (purple peaks) correlate well with CpG density (green overlay).
5-Methylcytosine (5-mC) antibody (mAb) tested by Methyl-DNA Immunoprecipitation (MeDIP). Methylated DNA derived from the promoter of the APC gene was amplified using 5me-dCTP and PCR. 25pg of methylated DNA was spiked into 500 ng of MseI digested human genomic DNA and subjected to the MeDIP procedure using 5μg of 5-Methylcytidine antibody or control mouse IgG. Real time quantitative PCR to amplify the methylated APC promoter DNA was performed on the immunoprecipitated DNA and results plotted as Fold Enrichment over control IgG.
5-Methylcytosine (5-mC) antibody (mAb) tested by dot blot analysis. DNA from the Methylated DNA Standard Kit (Catalog No. 55008) were spotted (5 ng per spot) on to a positively charged nylon membrane and blotted with 5-Methylcytosine antibody (2 ug/ml dilution). Lane 1: single-stranded unmethylated DNA. Lane 2: single-stranded DNA containing 5-methylcytosine. Lane 3: single-stranded DNA containing 5-hydroxymethylcytosine. Lane 4: double-stranded unmethylated DNA. Lane 5: double-stranded DNA containing 5-methylcytosine. Lane 6: double-stranded DNA 5-hydroxymethylcytosine.
Dot blot of 5-Methylcytosine (5-mC) antibody (rAb).Dot blot of double stranded DNA using 5-Methylcytosine (5-mC) antibody (RM231). The membrane was pre-spotted with 50, 5, and 0.5 ng/dot of double stranded 5-Hydroxymethylcytosine (5-hmC) DNA, 5-Methylcytosine (5-mC) DNA, and unmethylated DNA. The pre-spotted membrane was then blotted with 5-Methylcytosine (5-mC) antibody at 0.5 ug/ml.
MeDIP of 5-Methylcytosine (5-mC) antibody (rAb).MeDIP was performed using 5-Methylcytosine (5-mC) antibody (RM231) at a 2:1 DNA:Ab ratio. 1 ng of unmethylated, 5-Methylcytosine (5-mC) or 5-Hydroxymethylcytosine (5-hmC) DNA standard (897 bp) was spiked in 1 ug of genomic DNA isolated from HeLa cells as the control. Realtime PCR was then performed to determine the capture of DNA standard as in % of input.
Immunofluorescence stain of 5-Methylcytosine (5-mC) antibody (rAb).HeLa cells were stained using 5-Methylcytosine (5-mC) antibody (RM231) antibody at 1 ug/ml (red). Actin filaments have been labeled with fluorescein phalloidin (green), and nuclei stained with DAPI (blue).
Dot blot of AbFlex 5-methylcytosine antibody (rAb). DNA standards from Cat# 55008 (5, 1 and 0.2 ng as indicated) were spotted onto positively charged nylon membrane and blotted with AbFlex 5-methylcytosine recombinant antibody at a 1 ug/mL. Lane 1: Unmethylated DNA. Lane 2: DNA containing 5-methylcytosine. Lane 3: DNA containing 5-hydroxymethylcytosine.
AbFlex Ago1/2/3 antibody (rAb) tested antibody tested by immunoprecipitation. Active Motif miRNA IP Kit (Cat. No. 25500) was used with 293 cells (10 million cells per IP). Following IP using AbFlex Ago1/2/3 antibody (5 ug per IP) or a negative control IgG, qRT-PCR was performed on samples using primers for IGF2. Fold enrichment over the negative control IgG shows successful specific IP of IGF2 miRNA associated with the RISC complex.