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Rabbit anti m6a antibody

Manufactured by Synaptic Systems
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Sourced in Germany, United States

The Rabbit anti-m6A antibody is a research-use reagent designed to detect the presence of N6-methyladenosine (m6A) modifications in RNA samples. This antibody specifically binds to the m6A modification, allowing for the identification and analysis of m6A-containing RNA molecules.

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Lab products found in correlation

Hybond n membrane, by GE Healthcare (6 mentions) Superblock blocking buffer, by Thermo Fisher Scientific (4 mentions) Image studio software, by LI COR (4 mentions) Rnase inhibitor, by Thermo Fisher Scientific (4 mentions) Nanodrop 2000, by Thermo Fisher Scientific (4 mentions) Mirvana rna isolation kit, by Thermo Fisher Scientific (3 mentions) Dynabeads mrna purification kit, by Thermo Fisher Scientific (3 mentions) Dynabeads protein a, by Thermo Fisher Scientific (3 mentions) Total rnapure kit, by ZOMANBIO (2 mentions) Nebnext poly a mrna magnetic isolation kit, by New England Biolabs (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Ecl imaging system, by Bio-Rad (2 mentions) Trizol reagent, by Takara Bio (2 mentions) Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions) Horseradish peroxidase hrp conjugated anti rabbit igg, by Cell Signaling Technology (2 mentions) Non fat milk, by Bio-Rad (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Gnome xrq npc, by Syngene (1 mentions) Ecl chemiluminescence, by Merck Group (1 mentions)

20 protocols using rabbit anti m6a antibody

1

Quantifying mRNA m6A Modifications

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After being denatured by heating at 72°C for 5 min, mRNA in a volume of 1.5 μL was spotted on Hybond Nitrocellulose Membranes (Pall) and cross‐linked to the membrane by UV. One of the membranes was blocked using 5% non‐fat dry milk in TBST for 1 h at room temperature and inducted with rabbit anti‐m6A antibody (1:3000; Synaptic Systems) overnight at 4°C. Next day, the membrane was inducted with HRP‐conjugated goat anti‐rabbit IgG (1:5000) for 2 h at room temperature, then visualized on GNOME XRQ NPC (Syngene) using ECL chemiluminescence (Millipore). The other membrane was stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) for 2 h as the loading control.
Liu Y., Zhou T., Wang Q., Fu R., Zhang Z., Chen N., Li Z., Gao G., Peng S, & Yang D. (2022). m6A demethylase ALKBH5 drives denervation‐induced muscle atrophy by targeting HDAC4 to activate FoxO3 signalling. Journal of Cachexia, Sarcopenia and Muscle, 13(2), 1210-1223.
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2

Quantifying m6A Transcripts via RIP-qRT-PCR

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After verifying m6A binding with the anti-m6A antibody, anti-m6A RIP-qRT–PCR was used for quantification of m6A-modified MAT2A levels. The total RNA was extracted using the total RNApure Kit (#ZP404-1, ZOMANBIO, China) after METTL16 knockdown in HT22 cells. Then 18 μL of total RNA were subjected to fragmentation using Mg2+ fragmentation solution (#E6150SNEB, NEB, USA) at 94 °C for 3 min. Subsequently, RNA was purified again using a RNeasy micro kit (#74004, Qiagen, Germany) and quantified using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific). The 6 μg fragmented RNA suspended in 200 μL RIP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with 0.2 μL RNase inhibitor (#N8080119, Thermo Fisher Scientific) was subjected to RNA immunoprecipitation, in which 1 μg of a rabbit anti-m6A antibody (#202003, Synaptic Systems, Germany) conjugated with 8 μL of Dynabeads protein A (#10001D, Life technologies, USA) was used. After rotation at 4 °C for 2 h, the magnetic beads were washed five times with the RIP lysis buffer, and the precipitated RNA was eluted in the RIP lysis buffer containing 5 mg/mL m6A (#B5993, APExBIO, USA) at 55 °C for 30 min. The transcripts of interest contained in the input and immunoprecipitated RNA were quantified using qRT–PCR, as described above.
Zhang R., Zhang Y., Guo F., Huang G., Zhao Y., Chen B., Wang C., Cui C., Shi Y., Li S, & Cui H. (2022). Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA. Cell Death Discovery, 8, 432.
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3

Mapping of m6A Epitranscriptomic Landscape

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MeRIP-sequencing projects and subsequent data analysis were supported by Genesky Biotechnologies Inc. (Shanghai, China). Total RNA was isolated from SW480 cells transfected with shMETTL14-2 (shM14-2) or shControl (shCON), followed by poly (A) + RNA purification and fragmentation using NEBNext Poly (A) mRNA Magnetic Isolation Kit (New England Biolabs, UK). Concentration of RNA was detected on Nanodrop 2000 (Thermo Fisher Scientific, USA) and the integrality was guaranteed by Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Dynabeads Protein A (Thermo Fisher Scientific, USA) was mixed with rabbit anti-m6A antibody (Synaptic system, Germany) at 4 °C for 2 h in advance, then fragmented mRNA was incubated with the mixture for another 2 h to precipitate m6A-enriched RNAs. Qualified samples underwent Library Pooling and Sequencing using Illumina HiSeq 2500 machines. Following quality filter, the raw sequence data was mapped to human genome GRCh37/hg19 utilizing the HISAT2 software (v2.0.5) and the results were subjected to analyzed bioinformatically and statistically. The peak calling data and RNA-sequencing data were described in Supplementary Materials.
Wang H., Wei W., Zhang Z.Y., Liu Y., Shi B., Zhong W., Zhang H.S., Fang X., Sun C.L., Wang J.B, & Liu L.X. (2021). TCF4 and HuR mediated-METTL14 suppresses dissemination of colorectal cancer via N6-methyladenosine-dependent silencing of ARRDC4. Cell Death & Disease, 13(1), 3.
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4

m6A RNA methylation quantification

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Total RNA was extracted from the cells using Trizol reagent (TAKARA). mRNA was isolated and purified using Poly Attract mRNA Isolation System III with Magenetic Stand (Promega) following the manufacturer’s instructions. For m6A dot blot, mRNA was hybridized onto the Hybond-N + membrane (GE Healthcare). After crosslinking at 80 °C for 30 min, the membrane was blocked with 5% non-fat milk (Biorad) for 1 h, incubated with rabbit anti-m6A antibody (1:1000, Synaptic Systems, cat. No. 202003) at 4 °C overnight. Then the membrane was incubated with HRP-conjugated mouse anti-rabbit IgG (1:3000, Santa,sc-2357) at room temperature for 2 h. After being incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore), the immunocomplex was photographed using the ECL imaging system (Bio-Rad). Finally, the membrane was stained with 0.02% methylene blue to eliminate the difference in mRNA amount. Relative m6A level was quantified via gray intensity analysis using Image J.
Huang T., Liu Z., Zheng Y., Feng T., Gao Q, & Zeng W. (2020). YTHDF2 promotes spermagonial adhesion through modulating MMPs decay via m6A/mRNA pathway. Cell Death & Disease, 11(1), 37.
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5

RNA Quantification and Detection of m5C and m6A

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By using a NanoDrop, the concentration of the purified RNA was measured and diluted to 100 ng/μl and 50 ng/μl with RNase-free water. We denatured purified RNA by heating it to 95 °C for 3 min and cooling it on ice. Nucleic acid transfer was optimized using the Hybond-N + membrane (Solarbio). Incubation with mouse anti-m5C antibody (1:1000; MAb-081-010, Diagenode) or rabbit anti-m6A antibody (1:1000, 202003, Synaptic Systems) was performed overnight at 4 °C after UV crosslinking. All membranes were incubated with HRP-labelled mouse IgG secondary antibodies (ZB-2305, Zsbio Store-bio) or rabbit IgG secondary antibodies (ZB-2316, Zsbio Store-bio) for 1 h before visualization by an imaging system (Bio-Rad, USA). An ECL Western blotting Detection Kit (Sevenbio, Beijing, China) was used to visualize the membrane. The other membrane was loaded with methylene blue.
Huang F., Wang X., Zhong J., Chen H., Song D., Xu T., Tian K., Sun P., Sun N., Qin J., Song Y., Ma W., Liu Y., Yu D., Meng X., Jiang C., Xuan H., Qian D, & Cai J. (2023). Using integrated analysis from multicentre studies to identify RNA methylation-related lncRNA risk stratification systems for glioma. Cancer Cell International, 23, 156.
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6

Antibody-based Detection of RNA Modifications

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RNA Oligonucleotides were synthesized in-house with either m1A, m6A or A at a single internal position (5′-AC(m1A/m6A/A)UG-3′), spotted onto a nylon membrane (GE Healthcare) in decreasing amounts (1,000, 200, 40 and 8 pmol) and UV-crosslinked. Membranes were blocked with 5% non-fat dry milk in 1 × PBST (blocking buffer) for 1 h at 25 °C, and incubated overnight with either mouse anti-m1A antibody (1 μg ml−1, MBL) or rabbit anti-m6A antibody (1 μg ml−1, Synaptic Systems) in 1 × PBST at 4 °C. Following extensive washing with 1 × PBST, membranes were incubated with either HRP-conjugated goat anti-mouse IgG or anti-rabbit IgG antibody (1:2,500, Thermo Fisher Scientific) in blocking buffer for 1 h at 25 °C. Membranes were washed in 1 ×PBST, developed with ECL substrate (Thermo Fisher Scientific) and imaged with FluorChem imager (Protein Simple) or X-ray film. Competitive dot blots were performed on separate membranes spotted with 75 pmol of the m1A-containing oligonucleotide by co-incubation of anti-m1A antibody with increasing concentrations of either m1A or m6A competitor mononucleoside (0, 1, 2 and 4 μM).
Dominissini D., Nachtergaele S., Moshitch-Moshkovitz S., Peer E., Kol N., Ben-Haim M.S., Dai Q., Di Segni A., Salmon-Divon M., Clark W.C., Zheng G., Pan T., Solomon O., Eyal E., Hershkovitz V., Han D., Doré L.C., Amariglio N., Rechavi G, & He C. (2016). The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA. Nature, 530(7591), 441-446.
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7

m6A-RNA Immunoprecipitation and Sequencing

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Biological replicates of control and TARBP2 knockdown cells (MDA-LM2 background) were collected and processed as previously described (Alarcón et al., 2015b (link)). 1×107 cells per sample were lysed using LB1 buffer (50mM HEPES-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% Triton X-100) and 1X protease inhibitor cocktail (Thermo Scientific). The nuclear fraction was then lysed with M-PER buffer (Thermo Scientific) and diluted tenfold in dilution buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl) before the immunoprecipitation. Rabbit anti-m6A antibody (Synaptic Systems) and rabbit IgG control bound to protein A dynabeads (Invitrogen) were used for the immunoprecipitations. The immunoprecipitated RNA was eluted with N6-methyladenosine (Sigma-Aldrich), ethanol precipitated and resuspended in water. RNA was barcoded using ScriptSeq V2 kit (Epicentre) and sequenced at Rockefeller University Genomics Core.
Fish L., Navickas A., Culbertson B., Xu Y., Nguyen H.C., Zhang S., Hochman M., Okimoto R., Dill B.D., Molina H., Najafabadi H.S., Alarcón C., Ruggero D, & Goodarzi H. (2019). Nuclear TARBP2 drives oncogenic dysregulation of RNA splicing and decay. Molecular cell, 75(5), 967-981.e9.
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8

Dot Blot Analysis of m6A RNA Modifications

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Dot blots were performed as follows. Total RNA was isolated. The RNAs (100 and 250 ng respectively) were double diluted and spotted onto a nylon membrane (Sigma-Aldrich, GERPN1210B). Then the membranes were ultraviolet (UV) crosslinked and blocked in blocking buffer (5% milk in phosphate-buffered saline with 0.1% Tween 20) for 1 h. Rabbit anti-m6A antibody (Synaptic Systems, 202003) was diluted 1:1,000 and incubated with the membranes overnight (4°C). After washing twice with 0.1% phosphate-buffered saline-Tween 20, horseradish peroxidase conjugated anti-rabbit immunoglobulin G (Cell Signaling Technology, 7074S) was diluted 1:5,000 and incubated with the membranes for 1 h at room temperature. After extensive washing, membranes were detected with a 3,3′-diaminobenzidine peroxidase substrate kit (Yeasen Biotechnology, 36302ES01). The same amount of poly(A)+ RNAs were spotted on the membrane, stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) for 2 h, and washed with ribonuclease-free water for 5 h.
Song H., Feng X., Zhang H., Luo Y., Huang J., Lin M., Jin J., Ding X., Wu S., Huang H., Yu T., Zhang M., Hong H., Yao S., Zhao Y, & Zhang Z. (2019). METTL3 and ALKBH5 oppositely regulate m6A modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes. Autophagy, 15(8), 1419-1437.
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9

Quantifying Global m6A Levels in mRNA

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Total RNA was extracted from the peri-infarct ipsilateral cortical tissue using mirVana RNA Isolation Kit (Thermo Scientific) and purified with Dynabeads mRNA Purification Kit (Thermo Scientific). Global m6A levels in the mRNA were measured by dot blot analysis as described previously.25 Briefly, 40 ng of denatured mRNA was spotted onto Hybond N+ membrane (GE Healthcare), UV crosslinked, blocked with SuperBlock Blocking Buffer (Thermo Scientific) and probed with rabbit anti-m6A antibody (1:250, Synaptic Systems). Blots were visualized with enhanced chemiluminescence (ECL) and quantified using Image Studio software (LI-COR Biotechnology).
Chokkalla A.K., Mehta S.L., Kim T., Chelluboina B., Kim J.Y, & Vemuganti R. (2019). Transient focal ischemia significantly alters the m6A epitranscriptomic tagging of RNAs in the brain. Stroke, 50(10), 2912-2921.
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10

Quantitative m6A RNA Analysis

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HCT116 cells were treated with DMSO or CS1 at 290nM (IC50) for 48 h. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and poly (A)+ RNA was further enriched with PolyATract mRNA isolation System IV (Promega) according to the manufacturer’s instructions. The RNA samples were diluted in RNA binding buffer, and denatured at 65°C for 5 min. Then one volume of 20X SSC buffer was added into the RNA samples before dotted onto the Amersham Hybond-N+ membrane (GE Healthcare) with Bio-Dot Apparatus (Bio-Rad). The RNA samples were cross-linked onto the membrane via UV irradiation for 5 min. The membrane was stained with 0.02% methylene blue (MB) as loading control. After that, the membrane was washed with 1X PBS-T buffer, blocked with 5% nonfat dry milk and incubated with rabbit anti-m6A antibody (202003, Synaptic Systems, 1:2000 dilution) at 4°C overnight. Finally, the membrane was incubated with the HRP-conjugated goat anti-rabbit IgG (sc-2030, Santa Cruz Biotechnology) and developed with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare).
Phan T., Nguyen V.H., Su R., Li Y., Qing Y., Qin H., Cho H., Jiang L., Wu X., Chen J., Fakih M., Diamond D.J., Goel A, & Melstrom L.G. (2023). Targeting fat mass and obesity-associated protein mitigates human colorectal cancer growth in vitro and in a murine model. Frontiers in Oncology, 13, 1087644.
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