For Western Blotting, 100 mg of brain tissue was homogenized in RIPA (150 mM NaCl, 10 mM Tris (pH 7.2), 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, supplemented with protease inhibitor mixture and phosphatase inhibitor) and centrifuged at 10,000× g for 20 min at 4 °C. Then, 20–40 µg of proteins from total homogenates were resolved in 12% acrylamide gels for SDS-PAGE and transferred to Immobilon-FL membranes (Millipore, Burlington, MA, USA). NFκB (p65) (acetyl K310) (cat# ab19870) were diluted 1:1000 (v:v) in PBS with 1% BSA and 0.1% Tween 20. Mouse anti-GAPDH used at 1:20,000 (v:v) (clone 6C5 #MAB374 from Sigma-Aldrich) or Revert™ 700 Total Protein Stain for Western Blot Normalization (LI-COR) was used as a loading control. Secondary antibodies were diluted 1:20,000 (v:v) in 1% BSA and 0.1% Tween 20. Proteins were detected using the Odyssey Infrared Image System (LI-COR).
Revert 700 total protein stain for western blot normalization
Manufactured by LI COR
The Revert™ 700 Total Protein Stain is a fluorescent stain used for the normalization of Western blot signals. It provides a direct measurement of the total protein present in each lane, which can be used to normalize target protein signals.
Automatically generated - may contain errors
2 protocols using revert 700 total protein stain for western blot normalization
1
Western Blotting of Acetylated NF-κB
2
Puromycin Protein Quantification in Cell Lysates
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Microdissected cortices from the acute slices or N2a cells growing on a 10-cm culture dish or 5 × 106 LCLs following the puromycin treatment were lysed in sterile-filtered radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1% NP-40, and 0.25% sodium deoxycholate (SDC)] supplemented with 1× cOmplete protease inhibitor cocktail (Roche) by sonication 5× every 10 s. The lysates were cleared by centrifugation at 20,000g for 10 min at 4°C. Equal amounts of protein were resolved on 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted against puromycin and β-actin. Puromycin protein signal in the whole lane was normalized against β-actin to calculate the puromycin/β-actin ratio using ImageJ. Revert 700 Total Protein Stain for Western Blot Normalization (Licor) was used to show the equal protein loading.
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