The cell pellet (approximately 100 µL) was mixed gently by pipetting with an equal volume of pre-configured RIP Lysis buffer (RIP-12RXN, Sigma, USA) and incubated on ice for 5 min before storing at -80 °C. Subsequently, 50 µL of magnetic beads (20,164, Thermo, USA) were added to each centrifuge tube labeled with IP and Normal IgG. After adding 500 µL of RIP Wash Buffer to the tubes, they were briefly vortexed and centrifuged using a vortex mixer. Then, the centrifuge tubes were placed in the magnetic field to discard the supernatant. The magnetic beads were resuspended in 100 µL of RIP Wash Buffer in each tube, followed by the addition of 5 µg of antibody and thorough mixing. The tubes were rotated at room temperature for 30 min, followed by brief centrifugation and removal of the supernatant using the magnetic field. This washing step was repeated twice. Finally, 500 µL of RIP Wash Buffer was added to each tube, briefly vortexed, and placed on ice. The RNA Immunoprecipitation Kit (RIP-12RXN, Sigma, USA) was utilized following the instructions provided. RNA was purified using TRIzol (15,596,026, Sigma, USA), and cDNA was generated utilizing the mRNA reverse transcription kit with mRNA as a template. The resulting products were used for RT-qPCR experiments.
Rip 12rxn
Manufactured by Merck Group
Sourced in United States
The RIP-12RXN is a laboratory instrument designed for high-throughput RNA immunoprecipitation (RIP) experiments. It can simultaneously process up to 12 samples, enabling efficient and reproducible RNA-protein interaction studies.
Automatically generated - may contain errors
Lab products found in correlation
Ab200699, by Abcam (2 mentions)
Ago2 antibody, by Abcam (1 mentions)
Trypsin, by Merck Group (1 mentions)
Magnetic bead, by Thermo Fisher Scientific (1 mentions)
Trizol, by Merck Group (1 mentions)
Ab32381, by Abcam (1 mentions)
Ab186733, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
7 protocols using rip 12rxn
1
RNA Immunoprecipitation Protocol
2
Investigating miRNA-mRNA Interactions via RIP-qPCR
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RIP assay was performed using a RIP kit (cat. no. RIP-12RXN; Sigma-Aldrich; Merck KGaA). ESCs were digested with trypsin (Sigma-Aldrich; Merck KGaA) and collected the cells, and then cells were ransferred into a mixed solution containing PBS, nuclear separation buffer and double-distilled H2O. The solution was stirred on ice and centrifuged at 2,500 x g for 15 min at 4˚C. The precipitated nucleus of ESCs was resuspended in RIP buffer. The solution was uniformly divided into IgG and the Ago2 group. The supernatant in the IgG group was added to 10 µg anti-IgG antibody (cat. no. ab171870; 1:1,000; Abcam) while that in the Ago2 group was added to 10 µg Ago2 antibody (cat. no. ab186733; 1:30; Abcam), gently shaken and then incubated at 4˚C overnight. The supernatant was added to 40 µl protein A/G magnetic beads (cat. no. M2400; Beijing Solarbio Science & Technology Co., Ltd.) and cultured at 4˚C for 1 h. Unconjugated protein was removed by RIP buffer washing. The untreated cell lysate was used as an input group. The RNAs in the input group, IgG and Ago2 group were extracted and reverse-transcribed into cDNA. Subsequently, the expression of miR-191 and MIR503HG were measured via quantitative polymerase chain reaction (qPCR). Expression in the input group was considered to be positive control.
3
Investigating miRNA-Protein Interactions via RIP
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RNA-binding protein immunoprecipitation (RIP) was performed using an anti-AGO2 antibody (PB1030, BOSTER) and the RIP assay kit (RIP-12RXN, Sigma Aldrich) following the manufacturer’s protocol. Briefly, cells were collected and lysed using RIP lysis buffer. The cell lysates were then incubated with RIP buffer containing magnetic beads conjugated to the anti-AGO2 antibody or negative control IgG. The samples were incubated with proteinase K to digest proteins, and then the immunoprecipitated RNA was isolated. The purified RNA was subjected to qRT-PCR to detect the presence of miR-23a and circXPO1. The total RNA was used as the input control.
4
AGO2 Protein Binding to miRNAs
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5
RIP-Based Detection of miRNA-mRNA Interactions
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RIP kit (RIP‐12RXN, Sigma, Saint Louis, MO, USA) was performed to detect the interaction between miR‐144‐3p and ONECUT2 and between LncRNA TM1‐3P and miR‐144‐3p. Briefly, the cell precipitate lysis was added with equal volume RIP lysis, repeated blow, incubated on ice for 5 min, and stored at −80°C for later use. Magnetic beads were vortexed with a vortex oscillator, then a 900 μL RIP immunoprecipitation buffer was added. The cell lysis products were thawed quickly, and 100 μL supernatant was taken and added into the centrifuge tube containing 900 μL RIP immunoprecipitation buffer and magnetic beads. The products were rotated overnight at 4°C. At the same time, 10 μL cracking products were taken as the input tube. 10 μL cracking products were mixed with 2 × SDS loading buffer and incubated at 95°C for 5min. Then 500 μL RIP wash buffer was added, eddy briefly, and repeated cleaning six times. RNA was purified by proteinase K buffer. Finally, the expressions of miR‐144‐3p, ONECUT2, and LncRNA TM1‐3P were detected by qRT‐qPCR.
6
RNA Immunoprecipitation of MALAT1-miR-382-3p-BDNF-AGO2 Complex
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The RIP kit (RIP-12RXN, Sigma-Aldrich, USA) was adopted to detect the binding between MALAT1, miR-382-3p, BDNF, and AGO2 protein. Upon reaching 80–90% confluency, the culture medium was removed. Then, the cells were lyzed using an equal volume of RIPA lysis buffer (P0013B, Beyotime) for 5 min and centrifuged at 14,000 rpm for 10 min at 4 ℃, with the supernatant collected. A portion of the cell extract was used as input, and the rest was incubated with antibodies for co-precipitation (Zhang et al. 2020 (link)). The RIP antibody we used was AGO2 (1:100, ab186733, Abcam, UK) and IgG (1:100, ab200699, Abcam, UK, negative control).
7
Quantification of mRNA m6A Levels
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PASMCs were transfected with WTAP siRNAs or the control siRNA. Total RNA was extracted and processed using an RIP kit (Sigma, RIP-12RXN). Total m6A levels in mRNA were measured using an EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric) (Epigentek, NY, USA) according to the manufacturer's instructions.
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