Briefly, RNAs were extracted with a miRVana RNA extraction kit (Thermofisher). The RNAs were nanodropped and then spotted on the hybond N+ membrane (GE Healthcare) as previously described.19 (link) Equal amount of the RNA was spotted and then cross-linked by UV radiation for 10 min. After which, membranes were stained with methylene blue for 10 min. Methylene blue was rinsed off under running cold water, and the RNA images were taken; then, the blots were blocked in 10% milk in TBST for 1 h, and after which 1:1000 anti-5mC (Abcam) in BSA was incubated with the blot shaking overnight. Primary antibodies were removed, and we added the secondary polyclonal HRP (1:20,000) in TBST and incubated shaking for 2 h at room temp. The membranes were washed 6× in TBST, then developed with ECL (Cat No: 1,705,061), and band imaged on a Bio-Rad Chemi-doc imaging system.
Anti 5mc
Manufactured by Abcam
Sourced in United Kingdom
Anti-5mC is a laboratory tool used to detect the presence of 5-methylcytosine (5mC), a modified form of the DNA base cytosine. It is a specific antibody that can recognize and bind to 5mC, allowing for the identification and quantification of 5mC in various applications such as epigenetic studies.
Automatically generated - may contain errors
Lab products found in correlation
Anti 5hmc, by Active Motif (3 mentions)
Mirvana rna extraction kit, by Thermo Fisher Scientific (2 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Antih3k4m3, by Merck Group (1 mentions)
H3k27m3, by Merck Group (1 mentions)
Tissue dna purification kit, by EURx (1 mentions)
Sherlock ax isolation kit, by A&A Biotechnology (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Proteinase k, by EURx (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Anti glut3, by Abcam (1 mentions)
Anti tet2, by Cell Signaling Technology (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Positively charged nylon membrane, by Carl Roth (1 mentions)
α tubulin, by Merck Group (1 mentions)
Olig2, by Merck Group (1 mentions)
Pdgfa, by Santa Cruz Biotechnology (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
9 protocols using anti 5mc
1
Quantitative RNA methylation analysis
2
Quantifying DNA Modifications and Histone Marks
3
DNA Methylation Analysis Protocol
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Approximately 100 mg of the dermis was homogenized in the TissueLyser II (Qiagen, Hilden, Germany) in PBS at 4 °C, then digested with proteinase K (EurX, Gdańsk, Poland) for 16 h at 55 °C. DNA was isolated using the Tissue DNA Purification Kit (EurX). DNA was also isolated from primary fibroblast cultures using the Sherlock AX isolation kit (A&A Biotechnology, Gdynia, Poland).
DNA (100 ng) was denatured in 0.4 M NaOH and 10 mM EDTA for 10 min at 100 °C, neutralized with 2 M ammonium acetate, applied to a nitrocellulose membrane on the Bio-Dot instrument (Bio-Rad, Hercules, CA, USA), and UV-crosslinked. Dot-blots were blocked and then incubated with anti-5mC (1:2000, Abcam, Cambridge, UK) anti-5hmC, anti-5fC, or anti-5CaC antibodies (1:10,000, Active-Motif, La Hulpe, Belgium). Incubation with an anti-ssDNA antibody served as a loading control (1:1000, Enzo, New York, NY, USA). Next, the blots were washed in TBST and incubated with an appropriate secondary antibody. Signals were detected using the enhanced chemiluminescent (Bio-Rad) and GeneGnome Chemiluminescence imaging systems (Syngene, Cambridge, UK). The quantitative analysis was performed using Image Studio™ Lite software (LI-COR, Lincoln, NE, USA).
DNA (100 ng) was denatured in 0.4 M NaOH and 10 mM EDTA for 10 min at 100 °C, neutralized with 2 M ammonium acetate, applied to a nitrocellulose membrane on the Bio-Dot instrument (Bio-Rad, Hercules, CA, USA), and UV-crosslinked. Dot-blots were blocked and then incubated with anti-5mC (1:2000, Abcam, Cambridge, UK) anti-5hmC, anti-5fC, or anti-5CaC antibodies (1:10,000, Active-Motif, La Hulpe, Belgium). Incubation with an anti-ssDNA antibody served as a loading control (1:1000, Enzo, New York, NY, USA). Next, the blots were washed in TBST and incubated with an appropriate secondary antibody. Signals were detected using the enhanced chemiluminescent (Bio-Rad) and GeneGnome Chemiluminescence imaging systems (Syngene, Cambridge, UK). The quantitative analysis was performed using Image Studio™ Lite software (LI-COR, Lincoln, NE, USA).
4
Epigenetic Regulation via Ascorbic Acid
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l -Ascorbic acid was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA, A4544-25G). Primary antibodies, including anti-GLUT3, anti-5-mC and anti-5-hmC, were purchased from Abcam (Abcam, Cambridge, MA, USA). Anti-TET2 was purchased from Cell Signaling Technology (CST, Beverly, MA, USA).
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5
Quantifying 5mC and 5hmC DNA Modifications
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To detect 5mC and 5hmC levels, genomic DNA was extracted by phenol-chloroform-isoamyl alcohol, and the concentration of samples was serially diluted twofold. Dot blot analysis was performed as described previously [5 (link)]. Subsequently, genomic DNA was spotted on a positively charged nylon membrane (Roth, Karlsruhe, Germany), air dried for 15 min and cross-linked using the UV light (20 s, 1,200 J/cm2). Then membranes were blocked in 5% non-fat milk in PBS/0.1% Tween-20 for 1 h at RT before incubation incubated with anti-5mC (1:5,000; Abcam, UK) antibody or anti-5hmC (1:5,000; Active Motif, Carlsbad, CA, USA) overnight at 4°C followed by washing in PBS for 3 times. The membrane was then incubated with HRP-labeled anti-mouse/rabbit secondary antibody (1:5,000; Santa Cruz, USA) and signal was developed using an enhanced chemiluminescence reagent (Bio-Rad, USA). The intensity of signal was measured using Image J software and calibrated against the linear range of the standard curves to estimate the quantities of 5mC or 5hmC in each sample.
6
Antibody Validation for Western Blot and Immunohistochemistry
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The following primary antibodies were used for Western blots and immunohistochemical staining: Myc tag (Cell Signaling, 2272), PDGFA (Santa Cruz Biotechnology, sc-128), β-Actin (Sigma Aldrich, A1978), α-tubulin (Sigma Aldrich, T5168), wtIDH1 (Histobiotech, DIA-W09), muIDH1 (Histobiotech, DIA-H09), anti-Flag (Sigma Aldrich, F7425), anti-5-mC (Abcam, ab10805), and Olig2 (Millipore, AB9610).
7
Quantifying Global 5mC and 5hmC Levels
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To determine the global levels of 5mC and 5hmC in clinical tissues, 2 μl of DNA (200 or 400 ng/μl) from each sample was denatured and spotted onto a positively charged nylon-based membrane (Amersham Biosciences, Freiburg, Germany), which was cross-linked and blocked with 5% non-fat milk. After washing in 0.1% Tween 20 in PBS, the membrane was incubated with anti-5hmC (1:10,000, Active Motif, Carlsbad, CA) and anti-5mC (1:1000, Abcam, Cambridge, UK) overnight at 4°C followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, 1:5000). The signal was developed with WEST-ZOL Plus (Intron) and visualized using RAS-4000 (Fujifilm, Tokyo, Japan). Relative 5hmC intensity was calculated by dividing the positively stained areas by the total area using Multi Gauge v3.0 (Fujifilm). To estimate the relative concentration of DNA, we performed methylene blue staining (0.02% methylene blue in 0.3 M sodium acetate, pH 5.2).
8
Immunohistochemical Detection of 5hmC
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To examine the presence and distribution of the 5hmC signature in clinical tissues, FFPE sections were dewaxed and rehydrated through a graded series of ethanol followed by blocking of endogenous peroxidase activity for 15 min. Slides were then washed in PBS, and antigen retrieval was performed in a citrate buffer solution with microwaving for 10 min. Slides were then incubated with anti-5hmC (1:10,000; Active Motif) or anti-5mC (1:1000; Abcam) for 1 h at room temperature. Sections were then incubated with biotinylated secondary antibody and detected using the ChemMate Envision detection kit (Dako, Carpinteria, CA).
9
Detection of 5-Methylcytosine by Dot Blot
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Briefly, RNA were extracted with miRVana RNA extraction kit (Thermofisher). The RNA were nanodropped and then spotted on the hybond N+ membrane (GE Healthcare) as previously described (17) . Equal amount of the RNA were spotted and then cross linked by UV radiation for 10mins. After which membrane were stained with methylene blue for 10mins. Methylene blue were rinsed off under running cold water, and the RNA image were taken and the blot were blocked in 10% milk in TBST for 1hr and after which 1:1000 anti-5mC (Abcam) in BSA were incubated with the blot shaking overnight. Primary antibodies were removed and we added the secondary polyclonal HRP (1:20,000) in TBST and incubated shaking for 2hrs at room temp. The membrane were washed 6X in TBST and then developed with ECL (Cat No: 1705061) and band imaged on a Bio-Rad Chemi-doc imaging system.
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