Magna rip rna binding pretein immunoprecipitation kit

The binding of circ_0061265 and miR-885-3p to AGO2 protein was assayed according to the Magna RIP RNA-Binding Pretein Immunoprecipitation kit (Millipore, USA). After pre-cooled PBS washing, the cells were lyzed using an equal volume of RIPA lysis buffer (P0013B, Beyotime) in an ice bath for 5 min, and centrifuged at 14,000 rpm for 10 min at 4 °C followed by collection of the supernatant. Part of the cell extract was taken as input, and part was incubated with antibody for co-precipitation. Specifically, 50 µL magnetic beads were washed and resuspend in 100 µL RIP Wash Buffer, and 5 µg antibody was added for binding according to the grouping. Then, the magnetic bead-antibody complex was washed, resuspended in 900 µL RIP Wash Buffer, and incubated with 100 µL cell extract at 4 °C overnight. The sample was placed on the magnetic stand to collect the magnetic bead-protein complex. The sample and Input were digested with proteinase K and RNA was extracted for subsequent PCR detection. The antibody used in RIP was: AGO2 (ab32381, 1:50, Abcam, UK) and IgG (1:100, ab109489, Abcam, UK, NC).

The binding of NKILA and miR-195 with Argonaute 2 (AGO2) was detected using a Magna RIP RNA-Binding Pretein Immunoprecipitation kit (Millipore Inc., Bedford, MA, USA). The neurons were lysed with equal volume of radio-immunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime Biotechnology Co., Shanghai, China) on ice for 5 min, and then centrifuged at 14,000 rpm for 10 min at 4° C. Part of the cell extract was used as Input, while the remaining was probed with antibody for co-precipitation. The cell exact was incubated with rabbit polyclonal antibodies against IgG (ab109489, 1: 100, Abcam, Cambridge, UK; used as NC) or AGO2 (ab32381, 1: 50, Abcam, Cambridge, UK) for co-precipitation. In details, 50 μL magnetic beads of each system were resuspended with 100 μL RIP wash buffer (EHJ-BVIS08102, Xiamen Jiahui Biotechnology Co., Ltd., Xiamen, Fujian, China), followed by an incubation with 5 μg antibody according to experimental groups. After washing, the magnetic bead-antibody complex was resuspended in 900 μL RIP Wash Buffer and incubated with 100 μL cell exact at 4° C overnight. The samples were placed on the magnetic pedestals to collect bead-protein complexes. After detachment using proteinase K buffer, RNA was extracted for RT-qPCR analysis.