Quantifying Myc-tagged Protein Levels

Protein extraction, separation by electrophoresis, transfer to membranes, membrane incubation with antibodies and washes, and chemiluminescence detection were carried out as described (22 (link)). An anti-Myc tag polyclonal antibody (PA1-981; Thermo Fisher Scientific) and goat horseradish peroxidase–conjugated anti-rabbit IgG (H + L) (Thermo Fisher Scientific) were diluted at 1:2,000 and 1:10,000 and used as primary and secondary antibodies, respectively. The intensities of the bands corresponding to the Myc-tagged fusion proteins were quantified using ImageQuant TL software (GE Healthcare, Chicago, IL). These intensities were then utilized to normalize the relative expression levels of the PhERF1-regulated metabolic genes, which were determined through RT-qPCR analysis.

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Shoji T., Sugawara S., Mori T., Kobayashi M., Kusano M, & Saito K. (2023). Induced production of specialized steroids by transcriptional reprogramming in Petunia hybrida. PNAS Nexus, 2(10), pgad326.

Publication 2023

anti-Myc tag polyclonal antibody (PA1-981; goat horseradish peroxidase–conjugated anti-rabbit IgG (H + L) ImageQuant TL software

Anti antibody Anti igg Antibodies Chemiluminescence Electrophoresis Expression genes Goat Horseradish peroxidase Membrane Proteins Rabbit

Corresponding Organization :

Other organizations : University of Toyama, RIKEN Center for Sustainable Resource Science, University of Tsukuba

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Variable analysis

independent variables

Protein extraction, separation by electrophoresis, transfer to membranes, membrane incubation with antibodies and washes, and chemiluminescence detection

dependent variables

Intensities of the bands corresponding to the Myc-tagged fusion proteins
Relative expression levels of the PhERF1-regulated metabolic genes

control variables

Anti-Myc tag polyclonal antibody (PA1-981; Thermo Fisher Scientific) and goat horseradish peroxidase–conjugated anti-rabbit IgG (H + L) (Thermo Fisher Scientific) were diluted at 1:2,000 and 1:10,000 and used as primary and secondary antibodies, respectively

controls

Positive control: Not specified
Negative control: Not specified

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