The RIP kit was purchased from Thermo Fisher Scientific (Beijing, China). RIP lysis buffer and protease inhibitor cocktail were added to cell samples. The supernatant was collected by centrifugation. We mixed 200 μL supernatant and 800 μL lysis buffer as a RIP reaction system. AGO-specific antibody or corresponding IgG was added to the reaction system, respectively. After incubation at 4°C overnight, 20 μL Dynabeads Protein G was added. Beads were collected after incubation for 2 h at 4°C and washed five times. RNA was extracted using TRIzol reagent, reversed transcription by PrimeScript RT reagent Kit, and qPCR performed. The reaction conditions of qPCR were as follows: predenaturation at 95°C for 30 s and PCR amplification for 50 cycles at 95°C for 5 s and 60°C for 34 s.
Rip kit
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
The RIP kit is a laboratory equipment designed for the extraction and purification of RNA-binding proteins (RBPs) from cellular samples. It provides a reliable and efficient method to isolate and study RBPs, which play crucial roles in various biological processes.
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Lab products found in correlation
3 protocols using rip kit
1
RNA Immunoprecipitation (RIP) Assay
2
Quantify LINC00968 RNA Binding by RIP
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The anti-AGO RIP was performed using a RIP kit (Thermo Fisher Scientific, MA, USA). Briefly, the cell supernatants were incubated with protein A/G magnetic beads conjugated with anti-SNRNP70 antibody, normal rabbit IgG (Cat. #PP64B) or anti-AGO2 antibody (ab32381) at room temperature for 30 min. Anti-SNRNP70 antibody was used as a positive control, while normal rabbit IgG was used as a negative control. The RNA-binding Protein-RNA complexes (RIP) were incubated at 4 °C overnight. Subsequently, purified RNA was amplified by qRT-PCR to quantify LINC00968. Primers were specific to U1 snRNA. The primers used were as follows: U1: F: 5′-GGGAGATACCATGATCACGAAGGT-3′, R: 5′-CCACAAATTATGCAGTCGAGTTTCCC-3′, LINC00968 F: 5′-GCCCAGTTGACAGGAAATGT-3′, R: 5′-TTGGTTCTCAATGGGATGGT-3′, Homo-GAPDH: F: 5′-GGAGCGAGATCCCTCCAAAAT-3′, R: 5′-GGCTGTTGTCATACTTCTCATGG-3′.
3
RIP Assay for Ago2-bound RNAs
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Thermo Fisher RIP kit was acquired for performing RIP assay as guided by supplier (Thermo Fisher Scientific, Waltham, MA). In line with the user guide, cell lysates were conjugated with normal control IgG antibody (Millipore) or human Ago2 antibody (Millipore, Billerica, MA) in magnetic beads. The recovered RNAs were examined using qRT-PCR.
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