Cell lysis and Western blotting were performed as described previously [31 (link)] The following antibodies were used: a polyclonal rabbit anti-Hsp104 antibody (Thermo Scientific Project NJ1240S), anti-Pgk1 monoclonal antibody (Molecular Probes, Carlsbad, CA, USA), and rabbit anti-Hsp70 antibody (Enzo SPA 757). HPR conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Thermo Fisher, Waltham, MA, USA) and SuperSignal Plus Chemiluminescent Substrate (Thermo Scientific) were used to develop the Western blot. Amersham Imager 600 was used to scan the blot and the intensities were quantified using the ImageJ-win64.
Supersignal plus chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal plus chemiluminescent substrate is a laboratory reagent used for the detection and quantification of proteins in Western blot analysis. It generates a luminescent signal in the presence of the target protein, which can be measured using a luminometer or imager. The product provides a sensitive and reliable method for protein detection.
Automatically generated - may contain errors
4 protocols using supersignal plus chemiluminescent substrate
1
Protein Detection via Western Blotting
2
Quantification of Liver Protein Levels
3
Quantification of Liver Protein Levels
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Total liver proteins were extracted using radio-immunoprecipitation assay (RIPA) buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl (lauryl) sulfate (SDS) in phosphate buffered saline) supplemented with protease inhibitor cocktail (Bimake). Protein (30 μg) was separated on a SDS-PAGE gel and transferred to a PVDF membrane. Membranes were probed with appropriate primary and secondary antibodies and visualized with SuperSignal plus chemiluminescent substrate (Thermo Fisher Scientific). Densitometry analysis was performed with Image J and normalized to β-actin. Primary antibodies anti-FGF21 (# ab64857, Abcam) and anti-β-actin (#A-5441, Sigma) were used in this study.
4
Liver Protein Extraction and Western Blot
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Total liver protein lysates were extracted using radioimmunoprecipitation assay buffer (RIPA buffer containing 1% NP40, 0.1% sodium dodecyl (lauryl) sulfate, 0.5% sodium deoxycholate in phosphate-buffered saline) supplemented with protease inhibitor cocktail (Bimake). Plasma membrane fraction of liver tissues were extracted by using a MinuteTM plasma membrane and cell fractionation kit according to the manufacturer's instruction (Invent Biotechnologies, SM-005). Protein (30 μg) was separated on a SDS-PAGE gel and transferred to a PVDF membrane. Membranes were incubated with appropriate primary and secondary antibodies. SuperSignal plus chemiluminescent substrate was then used to visualize the results (ThermoFisher). Densitometry analysis was performed with the Un-Scan-It software and normalized to either β-actin or GAPDH for total protein or Sodium Potassium ATPase for plasma membrane protein. All the densitometry data were presented as mean ± SEM.
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