The EZ-Magna ChIP A/G kit (17–371; Millipore, Billerica, MA, USA) was applied in accordance with manufacturer’s instructions. After being sonicated, the cells were centrifuged at 12,000×g at 4 °C for 10 min to remove the insoluble components. The cells were then added with Protein G Agarose, incubated at 4 °C for 1 h and centrifuged at 5000×g for 1 min to remove the supernatant. Then 10 µL of supernatant was used as “Input” and the remaining supernatant was divided into two parts, which were further treated with H3K4me3 antibody (ab185637; 1:20, Abcam) and NC rabbit anti-human IgG (ab2410; 1:25, Abcam) at 4 °C using overnight incubation. Protein G Agarose was inverted and incubated at 4 °C for 1 h to precipitate the protein-DNA complexes. Following centrifugation at 5000×g for 1 min, the supernatant was discarded. The non-specific complex (protein-DNA complex) was eluted, and de-crosslinked at 65 °C. The recovered and purified DNA fragments were used as amplification templates for RT-qPCR experiments, and the enrichment of H3K4me3 in the LSD1 promoter region was detected by ChIP assay [10 (link), 12 (link)].
Ab185637
Manufactured by Abcam
Sourced in United States
Ab185637 is a laboratory equipment product. It is designed for general laboratory use. The core function of Ab185637 is to perform specific tasks required in a research setting. Detailed specifications and intended use are not available.
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Lab products found in correlation
5 protocols using ab185637
1
ChIP Assay for H3K4me3 Enrichment
2
Immunohistochemical Analysis of Tumor Tissues
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Tumor tissues were dissected, collected, and fixed in 10% phosphate-buffered formalin (1:10 tissue–formalin ratio) overnight at 4 °C. Then, Paraffin sections were performed according to standard protocols. Then, representative tumor areas of the paraffin-embedded specimens were incubated with the primary antibodies and the appropriate Alexa Fluor-coupled secondary antibodies.
Cells were fixed with 4% paraformaldehyde and permeablized with 0.5% Triton X-100 for 10 min, then blocked with 3% BSA for 1 h at room temperature (RT). Afterward, the cells were treated with their primary antibody overnight at 4 °C, rinsed three times with PBS, followed by incubating with secondary antibody for 1 h at RT. Subsequently, DAPI was used to label the cell nuclei. The following antibodies were used for immunohistochemistry (IHC) and immunofluorescence (IF): anti-KDM5A (3876, Cell Signaling Technology), anti-H3K4me3 (abcam, ab185637), and anti-Ki67 (abcam, ab15580). The results of IF were quantified using ImageJ software. The images of stained slides were photographed with a confocal fluorescence microscope (Olympus, FV3000, Japan).
Cells were fixed with 4% paraformaldehyde and permeablized with 0.5% Triton X-100 for 10 min, then blocked with 3% BSA for 1 h at room temperature (RT). Afterward, the cells were treated with their primary antibody overnight at 4 °C, rinsed three times with PBS, followed by incubating with secondary antibody for 1 h at RT. Subsequently, DAPI was used to label the cell nuclei. The following antibodies were used for immunohistochemistry (IHC) and immunofluorescence (IF): anti-KDM5A (3876, Cell Signaling Technology), anti-H3K4me3 (abcam, ab185637), and anti-Ki67 (abcam, ab15580). The results of IF were quantified using ImageJ software. The images of stained slides were photographed with a confocal fluorescence microscope (Olympus, FV3000, Japan).
3
ChIP Assay for SOX17 Promoter Analysis
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The ChIP assay was performed with a ChIP kit (MilliporeSigma) according to the manufacturer’s instruction. In brief, cells were treated with 1% formaldehyde for 10 min at room temperature to crosslink DNA and proteins. The DNA was then subjected to sonication (120 W, 15 cycles of 2 s on and 5 s off) to generate DNA fragments. The cell debris was then removed by centrifugation (13,000 × g at 4°C). The DNA-protein complexes in the supernatant were divided into 3 portions and incubated overnight at 4°C with rabbit anti-RNA polymerase II IgG, rabbit IgG, and rabbit anti-H3K4me3 IgG (ab185637, Abcam, 1:100), respectively. The next day, antibody-enriched DNA-protein complexes were precipitated by protein agarose and collected by brief centrifugation. The complexes were then subjected to washing and decrosslinking (65°C overnight), and the DNA fragments were then purified and amplified for SOX17 promoter detection via qRT-PCR.
4
ChIP-qPCR of H3K4me3 Modifications
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CHIP was performed using an EZ-Magna ChIP Chromatin Immunoprecipitation Kit (Millipore, USA), as previously described [25 ]. Briefly, CNE1cells were lysed, and the chromatin was mainly fragmented ranged from 300 bp to 900 bp by prior digestion with micrococcal nuclease and later ultrasonication. DNA/protein complexes were precipitated by overnight incubation with 4 μg antibodies against histone H3 tri-methylated lysine 4 (H3K4me3, ab185637, Abcam), and then incubated with magnetic protein A/G agarose beads for 2 h. After reversal of protein-DNA cross-links, the DNA was purified and then analyzed by qPCR. The primers for ChIP were as following: PKM promoter 0-0.5 k F: 5′-GGGCCAGACTGTTTCCTCTC-3′ and R: 5′-CTTTCTCCCAGGGCGACTTT-3′; PKM promoter 0.5–1 k F: 5′-GGAAGGAGAGAAGCTGGGGA-3′ and R: 5′-TCCGGCTTAAAGCGGTCATC-3′; PKM promoter 1–1.5 k F: 5′-GCTTTTCCTCTCCCCTGACC-3′ and R: 5′-TCAAAGGCAGGGAAGCAGAG-3′; PKM promoter 1.5 k–2 k F: 5′-CTCTTTTCTGCTGGGGAGGG-3′ and R: 5′-GACACCACCATAGCTCCCAC-3′.
5
Immunohistochemical Analysis of Histone Modifications
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Anti-H3.3antibody(#ab176840), anti-H3 antibody(#ab1971), anti-biotin antibody(#ab1227), anti-H3K4me3(#ab185637), and anti-H3K9me3(#ab8898) were purchased from Abcam (USA). GDNF was purchased from Peprotech (#450-44, USA). Male C57BL/6J mice were purchased from SIPPR-BK Animal Company (Shanghai, China).
The C18-4 cells was kindly provided by Prof. Zuping He, who was a principal investigator in Renji-Med X Clinical Stem Cell Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. The C18-4 cells were grown as described by He et al. [25 (link)].
The C18-4 cells was kindly provided by Prof. Zuping He, who was a principal investigator in Renji-Med X Clinical Stem Cell Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University. The C18-4 cells were grown as described by He et al. [25 (link)].
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