A portion of the cells collected for the BRET assay was used for Western blotting. Protein extracts (40 μg) from the transfected HEK293T/17 cells were loaded to commercially made 4–12% Bolt gel, separated by voltage potential using Mini Gel Tank (Thermofisher, Waltham, MA, USA), and transferred to polyscreen polyvinylidene difluoride transfer membrane (PerkinElmer, Waltham, MA, USA) using iBlot™ 2 Gel Transfer Device (Thermo Fisher Scientific, Waltham, MA, USA). Ponceau stain was used to demonstrate the equal loading of proteins in each well. Respective proteins were revealed by anti-RGS9 antibody (Proteintech, Rosemont, IL, USA, 17970-1-AP), anti-Gβ5 ATDG antibody [51 (link)], anti-R7BP antibody [29 (link)], and anti-GAPDH antibody (Millipore, Burlington, MA, USA, MAB374). Anti-Gβ5 antibody and anti-R7BP antibody were gifts from Dr. William Simonds (NIDDK, National Institutes of Health, Bethesda, MD, USA).
Polyscreen polyvinylidene difluoride transfer membrane
Manufactured by PerkinElmer
Sourced in United States
Polyscreen polyvinylidene difluoride transfer membrane is a laboratory equipment product used for the transfer of proteins, nucleic acids, or other macromolecules from electrophoresis gels to a solid support membrane. It is made of polyvinylidene difluoride material and serves as a support surface for the transferred molecules.
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2 protocols using polyscreen polyvinylidene difluoride transfer membrane
1
Western Blot Analysis of BRET Assay
2
Western Blot Protein Analysis
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Protein extracts (40 mg) were loaded, separated by voltage potential, and transferred to polyscreen polyvinylidene difluoride transfer membrane (PerkinElmer, Waltham, MA), as previously reported. 23 Respective proteins were revealed by goat antieIL-1b polyclonal antibody (AF-501-NA, 1:500; R&D Systems, Minneapolis, MN) or mouse antieb-actin monoclonal antibody (NB600-501, 1:4000; Novus Biologicals). Antibodies and markers used in this study are listed in Table 2.
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