hMSCs were lysed with RIPA buffer and 12 μg of proteins were loaded. SDS-PAGE (12% Bis-Tris gel, from Invitrogen, Thermo Fisher Scientific Inc.), Polyvinylidene difluoride transfer membrane and quantification BCA assay (Thermo Fisher Scientific Inc.) were used. Membranes were blocked in 5% milk for 1 h at room temperature and probed with the primary antibodies (anti-LC3B, anti-p62/SQSTM1 purchased from Sigma-Aldrich S.r.l.) (1:1000) overnight at 4 °C, followed by secondary antibodies (1:5000) for 1 h at RT. Bands were visualized using an enhanced chemiluminescence kit (SuperSignal™ West Pico PLUS, from Thermo Fisher Scientific Inc.). Quantification was assessed using Image Lab Software (Bio-Rad Laboratories S.r.l., Segrate, Italy).
Polyvinylidene difluoride transfer membrane and quantification bca assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
Polyvinylidene difluoride (PVDF) transfer membrane is a type of membrane used in Western blotting techniques to transfer proteins from a gel to a solid support for further analysis. The BCA (Bicinchoninic Acid) assay is a colorimetric detection and quantification method used to determine the total protein concentration in a sample.
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2 protocols using polyvinylidene difluoride transfer membrane and quantification bca assay
1
Western Blot Analysis of Autophagy Markers
2
Evaluating Cell Metabolism and Autophagy
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To evaluate cell metabolism, CellTiter-Blue R (Promega, Leiden, the Netherlands) assay was used. See Supplementary Materials for more information.
Western blot analyzes hMSCs were lysed with RIPA buffer and 12 μg of protein samples was loaded. SDS-PAGE (12% Bis-Tris gel, Invitrogen, CA, USA), Polyvinylidene difluoride transfer membrane and quantification BCA assay (Thermo Scientific, MA, USA) were used. Membranes were blocked in 5% milk for 1 h at room temperature and probed with the primary antibodies (anti-LC3B, anti-p62/SQSTM1 purchased from Sigma-Aldrich) (1:1000) overnight at 4 • C, followed by secondary antibodies (1:5000) for 1 h at RT. Bands were visualized using an enhanced chemiluminescence kit (SuperSignal™ West Pico PLUS, Thermo Scientific).
Western blot analyzes hMSCs were lysed with RIPA buffer and 12 μg of protein samples was loaded. SDS-PAGE (12% Bis-Tris gel, Invitrogen, CA, USA), Polyvinylidene difluoride transfer membrane and quantification BCA assay (Thermo Scientific, MA, USA) were used. Membranes were blocked in 5% milk for 1 h at room temperature and probed with the primary antibodies (anti-LC3B, anti-p62/SQSTM1 purchased from Sigma-Aldrich) (1:1000) overnight at 4 • C, followed by secondary antibodies (1:5000) for 1 h at RT. Bands were visualized using an enhanced chemiluminescence kit (SuperSignal™ West Pico PLUS, Thermo Scientific).
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