Total RNA was heated to 75℃ for 5 min, placed on ice for 1 min, and loaded onto Hybond-N + membranes. Membranes were crosslinked with 150 mJ/cm2 in a UV 254 nm Stratalinker 2400 (Stratagene). Then membranes were blocked with 5% nonfat milk in 0.1% Tween 20 PBS (PBST) for 1 h at room temperature and incubated with an anti-ac4C antibody (1:1,000, Abcam, ab253039, Cambridge, UK) in PBST at 4℃ overnight. The membranes were then washed three times with 0.1% PBST, probed with a HRP-conjugated secondary anti-rabbit IgG in PBST (1:1,000) at room temperature for 1 h, and washed three times with 0.1% PBST. Odyssey CLx and quantified with LI-COR Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA) was used to visualize the dots.
Anti ac4c antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-ac4C antibody is a laboratory reagent used in research applications. This antibody specifically recognizes and binds to the N4-acetylcytidine (ac4C) modification in RNA molecules. It can be used to detect and study the presence and distribution of ac4C in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Image studio software, by LI COR (1 mentions)
Ab253039, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Fusion fx imager, by Vilber (1 mentions)
Super ecl detection reagent, by Yeasen (1 mentions)
Hybond, by Cytiva (1 mentions)
Smarter stranded total rna seq kit v2, by Takara Bio (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Magnetic beads, by New England Biolabs (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
7 protocols using anti ac4c antibody
1
RNA Dot Blot for ac4C Detection
2
RNA dot blot analysis of ac4C
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RNA dot blotting was conducted using an anti‐ac4C antibody, as previously described, with few adjustments.
14 (link),
17 (link) Briefly, TRIzol was used to separate the RNA from cells. The Dynabeads™ mRNA Purification Kit was used to purify the mRNA and RNA was placed in denaturation solution (20X SSC solution: RNA = 1:1) at 95°C for 5 min, then quickly placed on ice for 2 min. RNA was then added to DEPC‐treated Amersham Hybond‐N+ membranes. Membranes were crosslinked six times at 150 mJ/cm2 in a UV 254 nm crosslinker and then immersed into methylene blue stain for 5 min. The stained membranes were photographed. Membranes were washed in 75% ethanol for 2 min and blocked for 1 h. Then, the membranes were incubated with an anti‐ac4C antibody (1:1000; Abcam) for 2 h at 25°C. Membranes were incubated with an HRP‐conjugated secondary antibody (1:2000; ProteinTech) for 1.5 h. After incubating the Super ECL Detection Reagent (Yeasen Biotechnology, Shanghai, China), the membranes were visualised using a FUSION FX imager (VILBER, France).
14 (link),
17 (link) Briefly, TRIzol was used to separate the RNA from cells. The Dynabeads™ mRNA Purification Kit was used to purify the mRNA and RNA was placed in denaturation solution (20X SSC solution: RNA = 1:1) at 95°C for 5 min, then quickly placed on ice for 2 min. RNA was then added to DEPC‐treated Amersham Hybond‐N+ membranes. Membranes were crosslinked six times at 150 mJ/cm2 in a UV 254 nm crosslinker and then immersed into methylene blue stain for 5 min. The stained membranes were photographed. Membranes were washed in 75% ethanol for 2 min and blocked for 1 h. Then, the membranes were incubated with an anti‐ac4C antibody (1:1000; Abcam) for 2 h at 25°C. Membranes were incubated with an HRP‐conjugated secondary antibody (1:2000; ProteinTech) for 1.5 h. After incubating the Super ECL Detection Reagent (Yeasen Biotechnology, Shanghai, China), the membranes were visualised using a FUSION FX imager (VILBER, France).
3
ac⁴C RNA Immunoprecipitation Protocol
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ac4C RNA immunoprecipitation was performed with several modifications from previous m6A-seq protocol (29 (link)). In brief, 4 μg of anti-ac4C antibody (Abcam) was prebound to 25 μl of Dynabeads protein G (Invitrogen) in PBS at room temperature for 1 hour, and then 4 μg of fragmented mRNA was spiked in with 4 pg of ac4C-positive and ac4C-negative probe and incubated with the antibody precoated protein G beads at 4°C for 4 hours in immunoprecipitation (IP) buffer [10 mM tris-HCl, 150 mM NaCl, and 0.1% (v/v) Igepal CA-630]. The RNA-antibody-beads complex was then washed with IP buffer for three times, ac4C-containing RNAs were lastly eluted with ac4CTP nucleoside. Both input and IP products were subjected to library construction using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara).
4
Quantifying Acetylated RNA Modifications
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To quantify the level of ac4C modification of the specific gene, acetylated RNA immunoprecipitation (acRIP) was performed and the procedure was similar to the m6A RNA immunoprecipitation (MeRIP) assay by replacing the anti-m6A antibody with an anti-ac4C antibody according to the manufacturer’s instructions (Millipore, USA). In a nutshell, the anti-ac4C antibody (Abcam) was mixed with the beads overnight. Then, the complex was incubated with the RNA samples. After eluting the RNA from the beads, qPCR was performed. Besides, the service of acRIP-seq in SW480 and DLD-1 cells was provided by Guangzhou Epibiotek Co., Ltd. (Guangzhou, China). The raw data could also be found in GEO with the number GSE210385.
5
RNA ac4C Immunoprecipitation and Quantification
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The RNA was bound with anti-ac4C antibody (1:50, rabbit, Abcam) and proteinase A
agarose affinity matrix overnight at 4°C. Moreover, 100 μg total RNA in
co-immunoprecipitation buffer (50 mm Tris HCl, 750 mm NaCl, and 0.5% Igepal
CA-630) containing RNase inhibitors were incubated at 4°C for 3 h, washed with
proteinase K containing Debuff (5 mm Tris·HCl, 1 mm EDTA, and 0.05% SDS) to
elute RNA from the beads. RNA ac4C was purified and extracted using
phenol/chloroform. RNA primers (Table S1 ) were designed and prepared and qPCR was performed.
agarose affinity matrix overnight at 4°C. Moreover, 100 μg total RNA in
co-immunoprecipitation buffer (50 mm Tris HCl, 750 mm NaCl, and 0.5% Igepal
CA-630) containing RNase inhibitors were incubated at 4°C for 3 h, washed with
proteinase K containing Debuff (5 mm Tris·HCl, 1 mm EDTA, and 0.05% SDS) to
elute RNA from the beads. RNA ac4C was purified and extracted using
phenol/chloroform. RNA primers (
6
Identifying ac4C-modified RNA in EV71
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RNA was extracted from EV71-infected Vero cells and concentrated viral supernatants using TRIzol reagent (Invitrogen) or transcribed by T7 polymerase. For acRIP, 300 μg total RNA, 10 μg supernatant RNA, or 10 μg in vitro-transcribed EV71 RNA was incubated with anti-ac4C antibodies (Abcam) or IgG antibodies (Proteintech, Rosemont, IL, USA) in 500 μl IP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris–HCl, pH 7.4) for 4 h at 4°C. The mixtures were incubated with 30 μl anti-rabbit antibodies conjugated with magnetic beads (NEB, Ipswich, MA, USA; cat. no. S1432S) for 2 h at 4°C and washed six times with 1 ml IP buffer. RNA was extracted using TRIzol reagent and quantified by qRT-PCR.
7
Detection of N4-Acetylcytidine in RNA
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Anti-ac4C dot blot assays were performed as previously described (20 (link)). Briefly, 5 μg RNA was denatured for 5 min at 75°C, placed on ice for 1 min, loaded onto Hybond-N+ membranes, and subjected to ultraviolet crosslinking. ac4C detection was carried out with anti-ac4C antibodies (Abcam, Cambridge, UK) using standard protocols. Signals were detected using a ChemiDoc MP imaging system (Bio-Rad Laboratories, Berkeley, CA, USA).
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