ChIP assays were performed as previously described (Cui et al., 2020 (link); Zhou et al., 2013 (link)) with 1 mg protein used for each immunoprecipitation reaction. The ChIP reaction was carried out with 2 μl WC-2 (Cheng et al., 2001a (link)), CPC-1, H2B (Abcam, ab1790), or H3ac (Millipore, 06-599) antibody. Immunoprecipitated DNA was quantified by real-time qPCR. Occupancies were normalized by the ratio of ChIP to Input DNA. ChIP was performed using 2 μl c-Myc monoclonal antibody (TransGen, HT101) to examine occupancy of Myc.GCN-5. Occupancies of ChIP were normalized using IgG. The primers used for ChIP-qPCR are shown in Key Resources Table.
Ht101
Manufactured by Transgene
The HT101 is a laboratory equipment product manufactured by Transgene. It is designed to perform high-throughput analysis tasks. The core function of the HT101 is to automate and streamline various laboratory procedures, enabling efficient data collection and processing.
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5 protocols using ht101
1
Chromatin Immunoprecipitation (ChIP) Assay Protocol
2
Co-Immunoprecipitation Assay for Protein Interactions
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Co-IP assays were performed as described previously (Lin et al., 2016 (link)) to detect protein–protein interactions. Total proteins were extracted from seedlings with extraction buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM DTT, 0.1% TritonX-100, and 1× complete protease inhibitor cocktail (Roche, 04693159001). Proteins were immunoprecipitated with antibody-conjugated agarose beads (20 μl) at 4°C for 2 h. Beads were washed three times with protein extraction buffer and eluted with 2× SDS loading buffer at 95°C for 5 min. Eluted proteins were detected by immunoblotting and analyzed with an anti-PIF4 (Huai et al., 2018 (link)), anti-phyB (Zhang et al., 2018 (link)), anti-SEU (Huai et al., 2018 (link)), anti-SIZ1 (Niu et al., 2019 (link)), anti-GFP, or anti-Myc antibody (TransGen, HT101).
3
Investigating OsRpp30 Acetylation in E. coli and Rice
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For the experiments in E. coli, GST‐OsRpp30 and His‐OsRpp30 expressed in E. coli, grown without or with 10 nm TSA, were first isolated by immunoprecipitation with glutathione (Abcam, #ab193267) and nickel (Qiagen, #30210) sepharose beads, respectively. In subsequent immunoblotting, the antibody against GST (TransGen, #HT601) or the poly‐His tag (TransGen, #HT501) was used to assess the amount of pulled down OsRpp30 fusion proteins, and a pan acetylation monoclonal antibody (Proteintech, Rosemont, IL, #66289‐1‐Ig) was used to detect OsRpp30 acetylation. For the experiment in rice protoplasts, the OsRpp30‐Myc plasmid alone or together with the HDT701‐Flag plasmid was transfected into protoplasts of the indicated genotypes. Subsequently, OsRpp30‐Myc was purified using anti‐Myc beads for use in immunoblotting. A Myc antibody (TransGen, #HT101) was used to assess OsRpp30‐Myc amount in each lane, and the acetylation antibody was used to determine OsRpp30‐Myc acetylation.
4
Co-Immunoprecipitation Assay Protocol
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Co-IPs were performed with the Dynabeads Protein G (Life Technologies, 10004D) according to the manufacturer’s instructions. Briefly, 1.5 mg Dynabeads was conjugated with antibodies or IgG overnight at 4 °C. Antibodies were used are: 10 μg IgG (Proteintech, B900610), or 10 μg anti-ADNP antibody (R&D Systems, A5919), or 10 μg anti-FLAG antibody (Proteintech, 20543-1-AP), or 10 μg anti-HA antibody (Abbkine, A02040), or 10 μg anti-MYC antibody (Transgen Biotech, HT101). The next day, total cell lysates and the antibody-conjugated Dynabeads were incubated overnight at 4 °C with shaking. After three-times washing with PBS containing 0.1% Tween, the beads were boiled at 95 °C for 5 min with the 6×Protein loading buffer and the supernatant was collected for future WB analysis.
5
Co-immunoprecipitation of rice Catalase Proteins
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For coIP assays, the full-length CDSs of OsCPK12, OsCATA, OsCATB, and OsCATC were separately amplified via PCR and fused with sequences encoding a GFP tag and an Myc tag driven by the 35S promoter. The constructs were coexpressed in rice protoplasts as described previously (He et al., 2018 (link)). Protoplasts were transfected and incubated for 14–20 h. Total proteins were extracted with NB buffer (50 mM Tris–MES [pH 8.0], 0.5 M sucrose, 1 mM MgCl2, 10 mM EDTA, and Roche plant protease inhibitor cocktail) and then immunoprecipitated with anti-MYC magnetic beads (Bimake, catalog no. B26301) according to the manufacturer’s instructions. Immunoprecipitated proteins were separated via SDS–PAGE (4%–25% gel) and analyzed by immunoblotting with anti-hemagglutinin (TransGen; HT301) or anti-Myc antibodies (TransGen; HT101). After incubation with a secondary antibody (HuaAn; HA1006) for 1.5 h, the immunoblot signal was visualized with Super ECL (Coolaber; SL1350). Primers used to generate the coIP constructs are listed in Supplemental Table 2 .
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