Total kernel protein was extracted by grounding in liquid nitrogen and suspending in extraction buffer (50 mM Tris-Cl, 2.5 mM EDTA, 150 mM NaCl, 0.2% NP-40, 20% glycerol, 1 mM PMSF and 1% plant cocktail [Sigma Aldrich]) on ice for more than 20 minutes. The lysate was then centrifuged at 12,000 rpm for 5 minutes, discarded the pellet, and centrifuged one more time. For co-immunoprecipitation, the protein lysate was incubated with 5 μl O2 antibody from guinea pig for 2 hours at least. Then the protein-antibody complex was incubated with 100μl ProteinA Sepharose CL-4B (GE Healthcare) for at least 30 minutes. After washing with extraction buffer for 4 times, western blot was carried out with O2 (1:1000 dilution) and ZmMADS47 (1:1000 dilution) antibodies.
Plant cocktail
Manufactured by Merck Group
Plant cocktail is a lab equipment product developed by Merck Group. It is designed to facilitate the preparation of nutrient solutions for plant growth and cultivation in controlled laboratory environments. The product provides a pre-formulated blend of essential minerals, vitamins, and other nutrients required for optimal plant development. Customers can utilize this product to create standardized growth media for a variety of plant species, supporting research and experimentation activities.
Automatically generated - may contain errors
4 protocols using plant cocktail
1
Protein Extraction and Co-Immunoprecipitation
2
Maize Kernel Protein Extraction and Analysis
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The total kernel protein (21 DAP) was extracted by grinding in liquid nitrogen and suspending in extraction buffer (50 mM Tris-Cl, 2.5 mM EDTA, 150 mM NaCl, 0.2% NP-40, 20% glycerol, 1 mM PMSF and 1% plant cocktail [Sigma Aldrich]) on ice for 20 min. The lysate was then centrifuged at 12,000 rpm for 5 min, the pellet was discarded, and the lysate was centrifuged one more time. A Superdex 200 10/300 GL Column (GE Healthcare) was first equilibrated in protein extraction buffer with an ÄKTA purifier system (GE Healthcare) until the UV baseline was smooth. A 500-μl volume of maize kernel lysate was injected into the system, and the flow speed was adjusted to 0.5 ml per minute. After the first volume (6 ml) flowed through, consecutive fractions of 500 μl each were then collected. The protein complex was then concentrated by acetone and analyzed by immunoblotting.
3
Extracting Total Protein from Seeds
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Preparation of total protein for immunoblotting from developing seeds and other tissues was previously described by Bernard et al. (1994) . Briefly, the tissues were homogenized in liquid N and then resuspended in equal volume of lysis buffer in 2-mL tubes (60 mM of Tris-Cl at pH 6.8, 1.5% [w/v] SDS, 4% [v/v] 2-mercaptoethanol, 10% [w/v] Suc, 1 mM of PMSF, and 1% plant cocktail [v/v]; Sigma-Aldrich). After 20 min of incubation on ice, the tubes were centrifuged at 12,000 rpm at 4°C for 15 min. The supernatant was transferred to new tubes for SDS-PAGE analysis. Zeins and non-zeins were prepared from mature maize seeds as previously described by Wallace et al. (1990) .
4
Co-immunoprecipitation of O2 and ZmRFWD3
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Total seed proteins were extracted by grinding in liquid N and resuspending the powder in extraction buffer (50 mM of Tris-HCl at pH 7.4, 2.5 mM of EDTA, 150 mM of NaCl, 0.2% [w/v] NP-40, 20% [v/v] glycerol, 1 mM of PMSF, and 1% plant cocktail [v/v]; Sigma-Aldrich) on ice for 20 min. The lysate was then centrifuged at 12,000g for 5 min at 4°C, and the supernatant was centrifuged one more time at 12,000g for 5 min at 4°C. For co-IP, the protein lysate was incubated with 5 mL of rabbit anti-O2 antibody for 2 h. Next, the protein-antibody complex was incubated with 100 mL of Protein A Sepharose CL-4B (GE Healthcare) for 1 h. After four washes with extraction buffer, the complexes were subjected to immunoblotting with anti-O2 (1:2,500 dilution) and anti-ZmRFWD3 (1:800 dilution) antibodies. The anti-O2 and anti-ZmRFWD3 antibodies were detected using goat antirabbit IgG conjugated to horseradish peroxidase at a dilution of 1:5,000 (cat. no. 12-348; Sigma-Aldrich).
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