RNA immunoprecipitation (RIP) assays were conducted using the Protein A/G Agarose Resin 4FF (YEASEN, Shanghai, China) following the manufacturer’s protocol. Briefly, cells were lysed by NP-40 lysis buffer (Beyotime, China). Then, 100 μl cell lysates were incubated with NP-40 buffer containing Protein A/G Agarose Resin 4F conjugated with human anti-Ago2 antibody (Cell Signaling Technology) at 4 °C overnight. After that, agarose beads were isolated by centrifugation and incubated with protease K to dissociate Ago2-RNA complex from the beads. The RNA fraction precipitated by RIP was analyzed by qRT-PCR.
Protein a g agarose resin 4ff
Manufactured by Yeasen
Sourced in China
Protein A/G Agarose Resin 4FF is a chromatographic media designed for the purification of antibodies. It consists of Protein A and Protein G immobilized on a 4% cross-linked agarose matrix. This resin provides a high binding capacity for a wide range of immunoglobulins from different species.
Automatically generated - may contain errors
2 protocols using protein a g agarose resin 4ff
1
RNA Immunoprecipitation (RIP) Assay
2
Immunoaffinity purification of GroEL
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Cells were lysed using a one-step bacterial active protein extraction kit (Sangon Biotech, China). The lysates were incubated with anti-GroEL antibody (Abcam, USA) at room temperature for 1 h. Protein A/G Agarose Resin 4FF (Yeasen, China) was extensively washed with washing buffer (0.15 M NaCl, 20 mM Na2HPO4, pH 7.0) and centrifuged at 500 rpm for 1 min. The lysate-antibody mixture was subsequently added to the washed beads, followed by incubation for 30 min at room temperature. The beads were then washed with washing buffer three times and eluted using 25 μL 1× SDS PAGE loading buffer33 .
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