Cells were harvested in NuPAGE™ LDS Sample Buffer (Invitrogen) for 10 min at 70°C and sonicated to shear genomic DNA. Protein samples were separated by electrophoresis using 4–12% Bis-Tris mini protein gels (Invitrogen) and transferred to Immun-Blot PVDF membranes (Bio-Rad #1620177). Blots were incubated with primary antibodies at 4°C and then with HRP conjugated secondary antibodies for 1 hour at room temperature. HRP was detected using ECL substrates (Thermo Scientific #34076) and Licor Odyssey Fc Imager.
Odyssey fc imager
Manufactured by Thermo Fisher Scientific
The Odyssey Fc Imager is a fluorescence imaging system designed for the detection and quantification of proteins, nucleic acids, and other biomolecules in a variety of applications. It utilizes high-resolution digital imaging and near-infrared fluorescence detection to provide sensitive and accurate results.
Automatically generated - may contain errors
3 protocols using odyssey fc imager
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of Protein Samples
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Cells were harvested in NuPAGE™ LDS Sample Buffer (Invitrogen) for 10 min at 70°C and sonicated to shear genomic DNA. Protein samples were separated by electrophoresis using 4–12% Bis-Tris mini protein gels (Invitrogen) and transferred to Immun-Blot PVDF membranes (Bio-Rad #1620177). Blots were incubated with primary antibodies at 4°C and then with HRP conjugated secondary antibodies for 1 hour at room temperature. HRP was detected using ECL substrates (Thermo Scientific #34076) and Licor Odyssey Fc Imager.
3
Western Blot Analysis of Protein Expression
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Cells were lysed in laemmli buffer with protease/phosphatase inhibitors and the lysate was boiled at 95 °C for 7 min. Lysates were run on 8% or 10% SDS gels and transferred onto nitrocellulose membranes. The membrane was blocked with 5% milk or Licor blocking buffer for 1 h at room temperature, then incubated with primary antibody overnight at 4 °C followed by secondary antibody for 1 h at room temperature. Blots were visualized using Licor Odyssey Fc Imager for two color or Thermofisher MyECL Imager for single color assays and analyzed on ImageStudio or ImageJ software respectively. Actin or GAPDH expression was used as loading control where indicated. One sample t-test was performed on normalized data for comparison of two groups. One-way ANOVA and post-hoc paired ratio t-test was performed on raw data for three or more groups. For degradation assays, K rate constant for the non-linear regression and slopes for linear regression were used to calculate statistical difference.
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