For in vitro pull-down assays, we cloned the full-length CDS sequence of COR27 in the pET28a vector and introduced the construct into Escherichia coli strain BL21 to produce recombinant COR27. We mixed 1 μg of purified His-COR27 fusion protein with 20 μL of His beads in His binding buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10% glycerol, and 1 mM PMSF). We incubated the mixtures at 4°C for 3 h. We then added 1 μg of purified MBP or MBP-COP1 fusion protein to the mixtures before an additional incubation of 1 h at 4°C. After four washes with binding buffer, we boiled the pellet fraction with 5× SDS protein loading buffer. We detected input and pull-down prey proteins by immunoblot analysis using anti-His (1:5000 [v/v], H1029-2ML; Sigma-Aldrich) and anti-MBP (1:5000 [v/v], cat. no. E8031S; New England Biolabs) monoclonal antibodies.
H1029 2ml
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The H1029-2ML is a lab equipment product from Merck Group. It is a 2 milliliter container designed for use in laboratory settings. The core function of this product is to provide a standardized storage and measurement solution for liquid samples or reagents.
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3 protocols using h1029 2ml
1
In Vitro Pull-Down Assay for COR27
2
Comprehensive Protein Extraction and Analysis
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For protein extraction, seedlings were frozen in liquid nitrogen, ground into powder, then resuspended in 2× SDS buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 1× cocktail of protease and phosphatase inhibitors, 1 mM PMSF). Samples were heated for 10 min at 65 °C, then centrifuged at 13,000 × g for 10 min at room temperature. The supernatants were transferred into new tubes, and the total protein concentrations were determined by the BCA method. Equal amounts of total proteins were separated in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred onto PVDF membranes. The subsequent immunoblots were performed as previously described52 (link). Antibodies used in this study were anti-HY5 (A gift from Rongcheng Lin’s lab, 1:1000 dilution), anti-BZR1 (A gift from Jianming Li’s lab, 1:1000 dilution), anti-Histone H3 (05-499, Millipore, 1:1000 dilution), anti-HSP (AbM51099-31-PU, Beijing Protein Innovation, 1:5000 dilution), anti-HA (H9658-.2 ML, Sigma-Aldrich, 1:2000 dilution), anti-Flag (F3165-.2MG, Sigma-Aldrich, 1:2000 dilution), Anti-phospho-GSK3 (Tyr279/Tyr216) (05-413, Millipore, 1:1000 dilution), anti-MBP (#E8031S, New England Biolabs, 1:5000 dilution), anti-His (H1029-.2ML, Sigma-Aldrich, 1:2000 dilution), and anti-GST (#2625, Cell Signaling Technology, 1:1000 dilution).
3
Protein Extraction and Western Blot Analysis
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Cell pellets corresponding to OD600 5–10 were protein extracted using the alkali extraction protocol described in [18 (link)], and samples corresponding to OD600 0.5–1 were analyzed by SDS-PAGE and western blotting (WB). Monoclonal mouse anti-His primary antibody (Sigma H1029-2ML, 1:5000 dilution) and anti-mouse-HRP secondary antibody (Santa Cruz Biotechnology sc-2060, 1:10,000 to 1:20,000 dilution), or polyclonal rabbit anti-NifU ([13 (link)], 1:5000 dilution), polyclonal rabbit anti-NifS ([13 (link)], 1:200 dilution), polyclonal rabbit anti-NifM (1:4000 dilution), and anti-rabbit-HRP secondary antibody (Sigma A0545-1ML, 1:10,000 dilution) were used.
After the immunodetection of proteins, polyvinylidene fluoride (PVDF) membranes were stained with Coomassie to determine the total protein loaded and the blotting efficiency, as described in [19 (link)].
Representative expression results are shown throughout the manuscript. For every figure, at least two transformant colonies were analyzed for each strain and the results were obtained from at least two biologically independent experiments.
After the immunodetection of proteins, polyvinylidene fluoride (PVDF) membranes were stained with Coomassie to determine the total protein loaded and the blotting efficiency, as described in [19 (link)].
Representative expression results are shown throughout the manuscript. For every figure, at least two transformant colonies were analyzed for each strain and the results were obtained from at least two biologically independent experiments.
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