MeIP was performed as detailed previously [40 (link)]. Briefly, genomic DNA was isolated from the HUVECs in the first instance, and then genomic DNA was sonicated to obtain 300–100 bp fragments. The denatured DNA fragments (800 ng) were incubated with 5-mC antibody (1:50; Abcam, Waltham, MA, USA) for 2 h at 4 °C and washed three times with IP buffer (10 mM NaPO4, 140 mM NaCl, 0.05% Triton X-100, pH 7.0), then Dynabeads (30 μL; Thermo, Waltham, MA, USA) were added into the mix with the DNA fragments and 5-mC antibody. The sample was then incubated in a rotating tube holder overnight at 4 °C. The DNA fragments bound with the 5-mC antibody were purified with Proteinase K and phenol/chloroform. The purified DNA fragments were then detected using real-time PCR to evaluate the DNA methylation level. The primer sequences for the real-PCR are listed in Table S1 .
5mc antibody
Manufactured by Abcam
Sourced in United States
The 5mC antibody is a laboratory reagent used to detect and quantify 5-methylcytosine, a modified DNA base that plays a role in epigenetic regulation. It is a highly specific antibody that can be used in various applications, including DNA methylation analysis, chromatin immunoprecipitation (ChIP), and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Dynabead, by Thermo Fisher Scientific (1 mentions)
Medip kit, by Abcam (1 mentions)
P3130, by Merck Group (1 mentions)
Alexa fluor 488 conjugated anti rabbit, by Jackson ImmunoResearch (1 mentions)
Ph2ax antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 conjugated anti mouse, by Jackson ImmunoResearch (1 mentions)
Smz1500, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
3 protocols using 5mc antibody
1
Methylation Enrichment Immunoprecipitation (MeIP)
2
Genome-wide DNA Methylation Analysis
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Genomic DNA was isolated by phenol–chloroform extraction as previously described. The MeDIP assay was carried out as recommended by the methylated DNA immunoprecipitation (MeDIP) kit (Abcam #ab117133). In brief, genomic DNA was sonicated to 200- to 600-bp. A total of 1 μg of the purified DNA fragments was used for the MeDIP reaction. MeDIP were performed with 1 µL of non-immune IgG or 1 µL of 5mC antibody (Abcam). The DNA was released by treatment with proteinase K and further purified for the library DNA preparation.
3
Epigenetic Modifications in Embryogenesis
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The embryos at the defined time points after fertilization were fixed by 4% polyformaldehyde overnight at 4°C. Then, they were dechorionated manually and dehydrated with methanol. The whole-mount IFs with Dnmt1 antibody (Santa Cruz Biotechnology, catalog no. sc-20701), 5mC antibody (Abcam, catalog no. ab10805), and pH2AX antibody [Cell Signaling Technology, catalog no. 2577S], were done with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, catalog no. D1306) staining and performed as previously described (20 (link)). The secondary antibodies were Alexa Fluor 488–conjugated anti-rabbit and Alexa Fluor 488–conjugated anti-mouse (Jackson ImmunoResearch; 1:200 dilution). After staining, embryos were deyolked by tweezers and mounted on glass slides in mounting medium (Sigma-Aldrich, catalog no. P3130) at animal polar upturned position. Images were acquired on 710 or 880 META laser scanning confocal microscope and manipulated by ZEN software. Treated or untreated embryos were anesthetized at desired stages with 0.02% tricaine and mounted in 5% methyl cellulose (Sigma-Aldrich, catalog no. M-6385) for observation, and phenotype pictures were taken under Nikon SMZ1500 microscope.
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