Protein was extracted with RIPA buffer supplemented with phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA), and protein concentration was spectrophotometrically measured by Bradford assay kit (Bio-Rad) according to manufacturer’s protocol. Then, 30–40 µg of protein were resolved by SDS-PAGE and transferred to Immobilon®-P PVDF membrane (Merck Millipore, Billerica, MA, USA) according to standard protocols. Membranes were incubated with primary antibody overnight at 4 °C, then washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (Amersham Biosciences) for 1 h at room temperature. Signals were detected with an ECL kit (GE Healthcare, Buckinghamshire, UK) and visualized and quantified using ChemiDoc MP Imaging system (Bio-Rad, Hercules, CA, USA). Protein levels were normalized by measuring total protein directly on the membrane using the criterion stain-free gel imaging system (Bio-Rad).
Criterion stain free gel imaging system
Manufactured by Bio-Rad
Sourced in United States
The Criterion stain-free gel imaging system is a device used for the detection and visualization of biomolecules, such as proteins and nucleic acids, separated on polyacrylamide gels. The system utilizes a stain-free technology that allows for the direct detection of proteins without the need for traditional staining methods.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Roche (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bradford assay kit, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Criterion tgx stain free gel, by Bio-Rad (1 mentions)
Sds page standard, by Bio-Rad (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
3 protocols using criterion stain free gel imaging system
1
Western Blot Protein Quantification
2
SDS-PAGE Analysis of FPI Samples
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The molecular weight (MW) distribution of the FPI samples with and without TG treatment were analyzed by SDS-PAGE performed under reducing conditions according to (Laemmli, 1970) (link). Precast 4-20% Criterion TGX Stain-Free gels (#567-8093, Bio-Rad, USA) with pre-stained SDS-PAGE standard (#161-0363, Bio-Rad, USA). The bands were visualized using a Criterion Stain Free™ Gel Imaging System (Bio-Rad, USA).
3
Protein Extraction and Visualization
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Total protein of different tissues was extracted and separated by SDS-PAGE. For additional details, see the Supplemental information. Subsequently, proteins were visualized by fluorescent detection after photo-activation (Criterion Stain Free gel imaging system, Bio-Rad). For immunostaining, proteins were transferred to a polyvinylidene fluoride membrane via semi-dry blotting using either a Trans-Blot Turbo (Bio-Rad) for spikes and nodes or iBlot system (Invitrogen) for aleurone extracted proteins. The blots were incubated overnight with monoclonal anti-GFP (3H9, 1/1000 dilution; ChromoTek GmbH) or polyclonal anti-SLR1 (1/5000 dilution; Cosmo Bio Co, Tokyo, Japan), followed by incubation with the appropriate alkaline phosphatase-conjugated secondary antibody. Detection of alkaline phosphatase reaction was performed according to the instruction manual until color development was complete (Bio-Rad), which could take 18-40 h.
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