The RIP assay was implemented via a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Bersinbio, Guangzhou, China). DU145 or LNCaP cells were lysed adopting RIP lysis buffer. Cell lysates were then divided into two equivalent parts for incubating with either anti-Ago2 antibody (ab32381, Abcam, Cambridge, MA, USA) or non-specific anti-IgG antibody (ab190475, Abcam). Magnetic beads (Invitrogen) were supplemented to cell lysates and incubation was continued for 1 h, after which were incubated with Proteinase K (Absin, Shanghai, China) for 1 h at 55 °C. Detection of the enriched RNA was subjected to RT-qPCR. Assay was undertaken in at least triplicate.
Proteinase k
Manufactured by Absin
Sourced in China
Proteinase K is a serine protease enzyme that is commonly used in molecular biology and biochemistry applications. Its primary function is to break down and digest proteins, making it a useful tool for various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Magnetic bead, by Thermo Fisher Scientific (4 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (3 mentions)
Anti ago2, by Abcam (2 mentions)
Rip lysis buffer, by Solarbio (2 mentions)
Ab190475, by Abcam (1 mentions)
Ab32381, by Abcam (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by BersinBio (1 mentions)
Anti ago2 antibody, by Merck Group (1 mentions)
Ez magna rip kit, by Merck Group (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti ago2 antibody, by Abcam (1 mentions)
Imprint rip kit, by Merck Group (1 mentions)
Control anti igg antibody, by Abcam (1 mentions)
6 protocols using proteinase k
1
Ago2 Enrichment via RIP Assay
2
Ago2-RIP Assay Protocol
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Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore) was applied to perform Ago2-RIP assay based on the protocol. Cells were reaped at a confluence of 80–90% and lysed in RIP lysis buffer with magnetic bead conjugated and anti-Ago2 antibody (Millipore) or IgG overnight at 4 °C. After digesting with proteinase K (Absin, Shanghai, China), the immunoprecipitated RNA was purified and analyzed by qRT-PCR.
3
RIP Assay for hnRNP U and SNRNP70
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According to the protocol of EZ‐Magna RIP kit (Millipore), RIP assay was carried out in SPC‐A1 and A549 cells. Cell lysates in RIP buffer were treated with magnetic beads conjugated to antibodies against hnRNP U, IgG as negative control or SNRNP70 as positive control. After treatment with proteinase K (Absin), qRT‐PCR was performed.
4
RNA-Binding Protein Immunoprecipitation Assay
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Using a Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA), RIP assay was carried out. After being harvested, transfected 5-8F or CNE-1 cells were lysed in RIP lysis buffer (Solarbio, Beijing, China). Subsequently, magnetic beads (Invitrogen) were added to the RIP lysis buffer (Solarbio) and then conjugated overnight with anti-Ago2 (Abcam) or anti-IgG (Abcam) at 4 °C. The immunoprecipitated RNA was acquired after digestion with proteinase K (Absin, Shanghai, China) and quantified by RT-qPCR.
5
Ago2 RIP Assay for RNA Binding
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An RIP assay was performed using a Magna RIP™ RNA Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) [6 (link)]. After the transfected MCF7 and MDA-MB-453 cells were harvested, the cells were lysed using RIP lysis buffer (Solarbio, Beijing, China). Next, we added magnetic beads (Invitrogen) to RIP lysis buffer (Solarbio) and then conjugated the beads with anti-Ago2 (Abcam) or anti-IgG (Abcam) (overnight, 4°C). After digestion with proteinase K (Absin, Shanghai, China) the immunoprecipitated RNA was acquired and then quantified by RT-PCR.
6
AGO2-RIP Assay for RNA Detection
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Imprint RIP kit (Millipore, Billerica, MA, USA) was adopted for RIP assay. RIP lysis buffer including protease inhibitor (Thermo Fisher Scientific) was used to lyse MHCC97-H, HCCLM3, and Hep 3B cells. Afterward, magnetic beads (Thermo Fisher Scientific) conjugated with anti-AGO2 antibody (Abcam) or control anti-IgG antibody (Abcam) were added. Proteinase K (Absin, Shanghai, China) was selected to digest the proteins in the immunoprecipitated complex. Finally, quantitative real-time RT-PCR was carried out to detect RNA in the complex. Experiments were conducted three times.
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