The RNA immunoprecipitation (RIP) assay was performed using the RiboCluster Profiler RIP assay kit (MBL), anti-TIA-1, anti-TIAR antibodies, and protein G sepharose beads (Cell Signaling) according to the manufacturers’ protocols and our previous study [22 (link)]. Cellular RNA was pulled down with the antibodies and pre-immune rabbit IgG (supplied with the kit). The co-precipitated RNA was purified and sequentially subjected to RT-qPCR to detect TPD52 and β-actin.
Protein g sepharose beads
Manufactured by Cell Signaling Technology
Sourced in United States
Protein G Sepharose beads are a solid-phase matrix used for the purification and isolation of antibodies. The beads are made of Sepharose, a cross-linked agarose gel, and are coated with Protein G, a bacterial protein that binds to the Fc region of immunoglobulins. This allows for the selective capture and recovery of antibodies from complex mixtures, such as cell culture supernatants or serum samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti tiar antibodies, by Cell Signaling Technology (2 mentions)
Protein g sepharose beads, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Protein g sepharose beads, by Santa Cruz Biotechnology (1 mentions)
Bca technique, by Beyotime (1 mentions)
Cas pr30011, by Proteintech (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using protein g sepharose beads
1
RIP Assay for Detecting RNA-Protein Interactions
2
Immunoprecipitation of ADK, SAHH, and His-tagged proteins
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HUVECs were washed twice with PBS and then were lysed with a RIPA buffer (Sigma, St. Louis, MO, USA) with 1% proteinase inhibitor cocktail (Roche, Basel, Switzerland) and 1% PMSF. After centrifugation of the cell lysates, the supernatant were preincubated for 1 h at 4 °C with 30 µl of protein G-sepharose beads (Sigma, St Louis, MO, USA) and then centrifuged to remove proteins that adhered nonspecifically to the beads and to obtain the target supernatant for the following IP experiment. protein G-sepharose beads were incubated with anti-ADK (1.5 µg per 500 µg of total protein) (sc-23360, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-SAHH (1.5 µg per 500 µg of total protein) (sc-292967, Santa Cruz Biotechnology, Dallas, Texas, USA) or anti-His (1:200, 2366, Cell Signaling Technology, Danvers, MA, USA) for 3–4 h. The antibody-conjugated protein G-sepharose beads and the target supernatant were incubated overnight. Immune complexes were isolated by centrifugation, washed 4 times with 0.05 M HEPES buffer, pH 7.1, containing 0.15% Triton X-100, 0.15 M NaCl, and 0.1 × 10-3 M sodium orthovanadate, and bound proteins were eluted by heating at 100 °C in loading buffer. Proteins were evaluated by immunoblotting.
3
RIPA Lysis and Immunoprecipitation
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Radioimmunoprecipitation assay (RIPA) lysate that had been previously refrigerated was used to lyse the transfected cells or tissue. The BCA technique was used to measure the concentration of total protein (Beyotime, China). SDS-PAGE was used to separate the protein lysates and the membrane was subsequently coated with PVDF. The primary antibody was incubated with the PVDF membrane at 4 °C overnight as per the manufacturer’s instructions. The PVDF membrane was then incubated for 2 h at room temperature with a goat anti-rabbit secondary antibody that was HRP-labeled (1:5000, CAS #pr30011, Proteintech, China), after which the bands were identified using a Tianneng Tanon-5200 automated chemiluminescence image analysis equipment (China). Cell lysates were combined with antibody and protein G-sepharose beads (Cell Signaling Technology) for immunoprecipitation at 4 °C for at least 6 h. The immunoprecipitated proteins were then heated at 95 °C for 5 min while being treated with 2X sampling buffer. The blots were cut prior to hybridisation with antibodies during blotting.
4
ChIP-qPCR Analysis of E Protein Binding to Zbtb16
5
RNA Immunoprecipitation Assay for TPD52
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The RNA immunoprecipitation (RIP) assay was performed using the RiboCluster Profiler RIP assay kit (MBL), anti-TIA-1, anti-TIAR antibodies, and protein G sepharose beads (Cell Signaling) according to the manufacturers' protocols and our previous study [22] . Cellular RNA was pulled down with the antibodies and pre-immune rabbit IgG (supplied with the kit). The co-precipitated RNA was purified and sequentially subjected to RT-qPCR to detect TPD52 and β-actin.
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