Washed cell pellets were resuspended in cold radioimmunoprecipitation assay buffer [150 mM NaCl, 1% Triton X-100 (v/v), 0.5% Na-deoxycholate (w/v), 0.1% SDS (w/v), 50 mM tris-HCl (pH 7.4), 50 mM NaF, and 2 mM EDTA] supplemented with 1× protease inhibitor cocktail (Sigma-Aldrich) and 1× PhosSTOP phosphatase inhibitor cocktail (Roche). Next, cells were incubated 30 min on ice with brief vortexing every 10 min. Following 2× 45-s sonication, the lysates were cleared (10 min, 20,000g, 4°C) and transferred into fresh tubes.

Homogenization of 25 mg of cardiac tissue samples in 400 μl of cold organ lysis buffer [50 mM Hepes (pH 7.4), 50 mM NaCl, 1% Triton X-100 (v/v), 0.1 M NaF, 10 mM EDTA, 0.1% SDS (w/v), 10 mM Na-orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor cocktail (Sigma-Aldrich), and 1× PhosSTOP phosphatase inhibitor cocktail (Roche)] was performed with the Precellys CK 14 (Bertin Technologies) (5000 rpm, 30 s). Cleared protein lysates (45 min, 20,000g, 4°C) were transferred into fresh tubes. Determination of protein concentration was performed with Bradford reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Protein lysates were stored at 80°C.

Protein samples were dissolved in SDS-PAGE loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue] before denaturation. Depending on the required range of protein sizes, the proteins were separated on 8 to 15% acrylamide gels [stacking gel: 5% acrylamide-bisacrylamide (37.5:1), 12.5 mM tris-HCl, 0.1% SDS (w/v), 0.25% Ammonium persulfate (APS), and 0.25% Tetramethylethylenediamine (TEMED) (pH 6.8); separating gel: 8 to 15% acrylamide-bisacrylamide (37.5:1), 37.5 mM tris-HCl, 0.1% SDS (w/v), 0.1% APS, and 0.1% TEMED (pH 8.8)] in running buffer [25 mM tris-HCl, 250 mM glycine, and 0.1% SDS (w/v) (pH 8.3)].

Transfer of proteins on a nitrocellulose membrane by Western blot was conducted in transfer buffer (30 mM tris-HCl, 240 mM glycine, 0.037% SDS, and 20% methanol) at 400 mA for 2 hours at 4°C. For a first evaluation of the transfer, shortly washed membranes (dH2O) were stained with Ponceau S solution (Sigma-Aldrich). Depending on the antibody requirements, destaining and blocking of membranes were performed for 1 hour either in 5% milk-PBST (Phosphate-Buffered Saline/Tween) or 3% BSA-TBST (Tris-Buffered Saline/Tween) on a gently shaking platform before subsequent immunodecoration with the indicated antibodies according to the manufacturer’s instructions. Secondary horseradish peroxidase–coupled antibodies (1:5000) were incubated for 1 hour before detection by Pierce ECL Western blotting substrate (Thermo Fisher Scientific). Densitometry-based quantification of Western blots was performed with ImageJ and Image Studio Lite Software.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.