For detection of the green fluorescent protein (GFP) fusion protein in RXR3 complementation plants, total protein was extracted as described previously (Ying et al., 2022 (link)). Equal amounts (30 µg) of each sample were separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred into Immobilon‐P PVDF membrane (0.45 μm; Millipore Sigma) and probed with 1/5000‐diluted monoclonal mouse anti‐GFP horseradish peroxidase (HRP)‐conjugated antibody (Miltenyi Biotec). Chemiluminescence detection was performed with ECL™ Prime Western Blotting System (Millipore Sigma) and UVP ChemStudio imager (Analytik Jena).
Protocol detail
Western Blot Analysis of GFP Fusion Proteins
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Antibody
Chemiluminescence
Green fluorescent protein
Horseradish peroxidase
Immobilon p
Membrane
Mouse
Plants
Protein
Pvdf
Sds page
Corresponding Organization : Noble Research Institute
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Variable analysis
independent variables
- None explicitly mentioned
- Detection of green fluorescent protein (GFP) fusion protein in RXR3 complementation plants
- Equal amounts (30 µg) of each sample
- Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE)
- Immobilon‐P PVDF membrane (0.45 μm; Millipore Sigma)
- 1/5000‐diluted monoclonal mouse anti‐GFP horseradish peroxidase (HRP)‐conjugated antibody (Miltenyi Biotec)
- Chemiluminescence detection with ECL™ Prime Western Blotting System (Millipore Sigma) and UVP ChemStudio imager (Analytik Jena)
- No positive or negative controls were explicitly mentioned in the input protocol.
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