Whole cell protein extracts were prepared by cell lysis with SDS-PAGE buffer (80 mM Tris-HCL pH 6.8, 16% glycerol, 4.5% SDS, 450 mM DTT, 0.01% bromophenol blue) with 200 U/mL benzonase (Sigma-Aldrich, St. Louis, MO, USA), 50 μM MgCl2 and were boiled for 5 min. Equal amounts of protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After 1 h blocking with 5% non-fat dry milk in 1× PBS, 0.1% Tween20 at room temperature, membranes were incubated with antibodies as follows: anti-DNMT1 (2 µg/mL, Active Motif, Carlsbad, CA, USA), anti-DNMT3A (1:1000, Cell Signaling), anti-H3K36me3 (1:500, Abcam, Cambridge, UK), α-tubulin (1:15,000, Sigma-Aldrich) and anti-histone H3 (1:1000, Abcam). Detection was performed with the appropriate secondary antibodies (Bio-Rad, Hercules, CA, USA) and enhanced luminescence substrate (Pierce ECL, Thermo Fisher Scientific, Waltham, MA, USA). Details of antibodies used are mentioned in Table S6 .
Anti dnmt1
Manufactured by Active Motif
Sourced in United States
Anti-DNMT1 is a lab equipment product that serves as an antibody targeting the DNA methyltransferase 1 (DNMT1) protein. DNMT1 plays a crucial role in maintaining DNA methylation patterns during cell division. This antibody can be used for various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and study the DNMT1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Benzonase, by Merck Group (1 mentions)
Anti h3k36me3, by Abcam (1 mentions)
Anti dnmt3a, by Cell Signaling Technology (1 mentions)
Dnmt3a, by Active Motif (1 mentions)
Branson s250 digital sonicator, by Emerson (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Itaq polymerase, by Bio-Rad (1 mentions)
H3k36me3, by Active Motif (1 mentions)
H3k27me3, by Active Motif (1 mentions)
H3k4me3, by Active Motif (1 mentions)
Anti h3k9me3, by Active Motif (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Chemiluminescence imaging system, by Bio-Rad (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Anti snail, by Cell Signaling Technology (1 mentions)
3 protocols using anti dnmt1
1
Western Blot Protein Extraction and Analysis
2
ChIP-seq Protocol for Epigenetic Profiling
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Chromatin immunoprecipitation (ChIP) was performed as previously published (6) . Briefly, cells were treated with 1% formaldehyde, then 0.125 M glycine was added. The cells were collected in PBS, resuspended in lysis buffer and sonicated (BRANSON S250 digital sonicator, Branson, Danbury, CT, USA). Pre-cleared chromatin was quantified using Qubit (dsDNA BR Assay Kit, Life Technologies); 2 ug chromatin was incubated overnight at 4°C with anti-IgG (Santa Cruz Biotechnology, Dallas, TX, USA), anti-DNMT1, -DNMT3A, -DNMT3B, and anti-H3K9me3, -H3K27me3, -H3K4me3 and -H3K36me3 (all Active Motif). Ten percent of the total lysate was used for input control. DNA was extracted with the Chromatin IP DNA extraction kit (Active Motif) following the manufacturer protocol.
Immunoprecipitation products were amplified using iTAq Polymerase (Bio-Rad, Hercules, CA, USA) and specific primers (Supplementary Table 1B).
Immunoprecipitation products were amplified using iTAq Polymerase (Bio-Rad, Hercules, CA, USA) and specific primers (Supplementary Table 1B).
3
Western Blotting Protein Expression Analysis
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Cell pellets were lysed in RIPA buffer containing 1 mM PMSF and 1% complete protease inhibitor cocktail (Roche Applied Science; Indianapolis, IN, USA). After quantification, equal amounts of lysate were separated by SDS-PAGE and then transferred onto NC membranes (Pall Life Science, Port Washington, NY, USA). Membranes were blocked in 5% nonfat milk and incubated with anti-DNMT1(1:1000, Active Motif; Carlsbad, CA, USA), anti-E-cadherin(1:1000, Cell Signaling Technology; Beverly, MA, USA), antivimentin(1:1000, Cell Signaling Technology; Beverly, MA, USA), anti-Snail(1:400, Cell Signaling Technology; Beverly, MA, USA), and anti-β actin (1:1000, Cell Signaling Technology; Beverly, MA, USA) primary antibodies, followed by incubation with goat-anti-rabbit or goat-anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (1:2000, Pierce; Rockford, IL, USA). The protein signals were visualized using enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA) by a chemiluminescence imaging system (Bio-Rad, Richmond, CA, USA).
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