HepG2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Anti-β-actin antibody (clone AC-15) and CoCl2 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rabbit anti-human HIF-1α antibody was purchased from Abcam (Cambridge, MA, USA). Pierce™ ECL Western Blotting substrate and goat anti-rabbit immunoglobulin G (IgG) antibody conjugated to horseradish peroxidase (HRP) were purchased from Pierce (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Polyvinylidene fluoride (PVDF) membrane was purchased from EMD Millipore (Billerica, MA, USA). Rabbit anti-5-hmC antibody was purchased from Active Motif (Carlsbad, CA, USA). Dulbecco's modified Eagle's medium (DMEM) (REF11965), fetal bovine serum (FBS) (REF16000), trypsin/EDTA, DAPI and donkey anti-rabbit IgG antibody conjugated to Alexa Fluor 594 were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).
Rabbit anti 5hmc antibody
Manufactured by Active Motif
Sourced in United States
The Rabbit anti-5hmC antibody is a primary antibody that specifically recognizes 5-hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine. This antibody can be used to detect and study the presence and distribution of 5hmC in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cocl2, by Merck Group (1 mentions)
Anti β actin antibody clone ac 15, by Merck Group (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Protein g conjugated magnetic beads, by Thermo Fisher Scientific (1 mentions)
H3k27me3, by Abcam (1 mentions)
H4k12ac, by Abcam (1 mentions)
Rat anti brdu antibody, by Abcam (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
H3k4me3, by Abcam (1 mentions)
H3k9me2, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
Red x conjugated mouse anti rabbit monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Clariostar plus, by BMG Labtech (1 mentions)
Aec staining kit, by Merck Group (1 mentions)
6 protocols using rabbit anti 5hmc antibody
1
Hypoxia-Induced HIF-1α Signaling
2
Hydroxymethylated DNA Immunoprecipitation Assay
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PBT003 cells were transduced with relevant shRNA-expressing lentivirus. Three days after, hydroxymethylated DNA immunoprecipitation assay was performed using 5 million cells and 1 μl rabbit anti-5hmC antibody (Active Motif; Catalogue # 39770)51 (link) for each reaction. The immunoprecipitation was carried out using magnetic beads conjugated protein G (Thermo Fisher). Primers used for BTG and PPP2R1B RT-PCR are listed in Supplementary Table 1 .
3
Embryonic DNA Epigenetic Modifications
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5′-Methylcytosine (5mC), hydroxymethylcytosine (5hmC) and BrdU were detected in embryonic DNA following fixation in 4% (w/v) paraformaldehyde and treatment with 2 M HCl for 30 min. Fixed embryos were processed as soon as possible but were stored where necessary at 4 °C. For primary antibody labelling, samples were incubated overnight at 4 °C with mouse anti-5mC antibody (1:200 (v/v); EMD Millipore, UK), for 1.5 h at 37 °C with rabbit anti-5hmC antibody (1:200; Active Motif, USA) or for 1.5 h with rat anti-BrdU antibody (1:100; Abcam, UK). Additional primary antibodies recognized Oct4 (1:100; Santa Cruz, USA), Cdx2 (1:100; BioGenex Laboratories, USA), H3K4me3 (1:250; Abcam, UK), H3K9me2 (1:50; Abcam), H3K27me3 (1:50; Abcam) and H4K12ac (1:250; Abcam). Primary antibody incubation was followed by a 1 h incubation at 37 °C with the appropriate secondary antibody (1:250; Life Technologies, UK) conjugated to Alexa 350, Alexa 488 and/or Alexa 594. DNA was stained by incubating samples at 37 °C for 20 min in propidium iodide (1:200; Sigma, USA) or Hoechst 33342 (1:1,000; Sigma).
4
Quantitative 5-Hydroxymethylcytosine Imaging
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1 × 105 cells were cultured on a cover glass in a 12-well plate with 700 ul of medium. The cells were allowed to grow to desired morphology and density before staining procedure. To stain the cells, cells were first washed once with phosphate buffered saline (PBS) and fixed by 4% paraformaldehyde/4%sucrose in PBS at room temp, followed by permeabilization and DNA denaturation by 0.2% TritonX-100 in 4M HCl. After that, the cells were washed with PBS and blocked in 80 μl BSA (3%). The cells were incubated with rabbit anti-5hmC antibody (1:500, Active Motif) in BSA (3%) at 4°C overnight, and then conjugated with RED-X-conjugated mouse anti-rabbit monoclonal antibody (1:500, Santa Cruz) and DAPI (1:1000, Santa Cruz). The glass slides were mounted with a cover slip before imaging.
5
Enzymatic Assay for DNA Hydroxymethylation
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TET enzymatic activity was measured using an enzyme-linked immunosorbent assay–based plate assay for DNA hydroxymethylation essentially as described in (88 (link)). Briefly, the wild type or mutated version of Drosophila TET CD was cloned in pET28a expression vector, and the corresponding His-tagged protein was overexpressed in Escherichia coli BL21 (DE3) CodonPlus RIL cells and purified on nickel–nitrilotriacetic acid agarose beads as indicated in (41 (link)). Recombinant TET protein (2 μM) was incubated with 400 ng of biotin-labeled double-stranded DNA substrate [prepared by PCR with 5-methyl-dCTP (2'-deoxy-5-methylcytidine 5'-triphosphate) (5mdCTP) instead of dCTP to methylate all its cytosines] in reaction buffer [50 mM Hepes (pH 6.8), 100 μM ammonium ion(II) sulfate hexahydrate, 1 mM α-ketoglutarate, 1 mM ascorbic acid, and 50 mM NaCl] at 37°C. Reaction was stopped at different time points by adding NaOH, and biotinylated DNA substrate was incubated in an avidin-coated 96-well plate (Sigma-Aldrich) for 1.5 hours. Rabbit anti-5hmC antibody (1:10,000, Active Motif) and goat anti-rabbit horse radish peroxidase (HRP)–conjugated secondary antibody (1:5000, GE Healthcare) were used to measure 5hmC levels by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific) on a CLARIOstarPlus (BMG Labtech).
6
Immunohistochemical Profiling of Tumor Markers
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5 μm-thick tissue slices were sectioned from formalin-fixed paraffin-embedded surgically resected tumors and mounted onto polylysine coated slides. Before staining, tissue slides were sequentially deparaffinized and rehydrated using xylene solution and ethanol series. Heat-induced epitope retrieval was carried out in boiling Tris-EDTA buffer (pH 9.0) for 20 minutes, followed by thorough wash with 0.025% Triton X-100 buffer. Blocking was conducted in TBS buffer containing 10% goat serum and 1% BSA. Slides were incubated overnight in a humid 4°C chamber with 1:200 diluted rabbit anti-MBD3 antibody (Pierce) or 1:500 diluted rabbit anti-5hmC antibody (Active Motif). After blocking endogenous peroxidase with 0.3% H2O2, goat anti-rabbit IgG-HRP conjugates (Life technologies) were applied for 1 hour at room temperature. Color signal was developed by using AEC staining Kit (Sigma) and then the slides were sealed in mounting medium prior to imaging.
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