The protein samples were prepared using radioimmunoprecipitation assay lysis and extraction buffer (catalog no. 89900, Thermo Scientific, IL, USA), sample buffer, protease inhibitor cocktail (Roche, Basel, Switzerland), and dithiothreitol. Next, the samples were heated for 5 min at 96°C. Protein quantitation was done using a bicinchoninic acid protein assay kit (Pierce, Appleton, WI, USA). A549 cell protein extracts (10 μg/lane) were separated on 12% polyacrylamide gels (Invitrogen; Thermo Fisher Scientific, Inc.), transferred to nitrocellulose membranes (GE Healthcare) at room temperature for 1 h, and then incubated in 2% blocking reagent (GE Healthcare) in tris-buffered saline buffer containing Tween 20 (0.1%) (TBST) overnight at 4°C. Next, the membranes were incubated in 5% blocking buffer containing diluted primary antibody (Supplementary Table 1 ) at 4°C overnight. The membranes were washed and incubated with a secondary antibody near-infrared system (LiCOR, USA) at a 1: 10 000 dilution in TBST at room temperature for 90 min. The membranes were imaged (LI-COR Odissey Clx Western Blot Imager, USA), and the intensity of the bands was quantified using LiCOR quantification software.
Blocking reagent
Manufactured by GE Healthcare
2% blocking reagent is a laboratory solution used to reduce non-specific binding in immunoassays and other protein-based experiments. It is a ready-to-use formulation that effectively blocks unoccupied binding sites, minimizing background signals and improving the accuracy of target analyte detection.
Automatically generated - may contain errors
2 protocols using blocking reagent
1
Western Blot Protein Analysis
2
Western Blot Analysis of Muscle Proteins
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The dissected soleus muscle was weighted, homogenised in lysis buffer containing 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and protease inhibitors. After centrifugation, protein samples were heat denatured at 96°C for 5 minutes. Samples (10 μg/lane) were separated by SDS-PAGE and were then transferred to a nitrocellulose membrane (GE Healthcare) for 1 hour at room temperature and blocked overnight at 4°C in 2% blocking reagent (GE Healthcare) in Tris-buffered saline buffer with 0.1% Tween 20. Immunoblotting was performed using a mouse monoclonal anti-mTOR (#2972); AKT (#9272), p-mTOR (#2971), LC3 (#12741), p62 (#5114), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) thermo scientific AM4300 were purchased from Cell Signalling Co., Ltd.) with dilution 1:1000. The signals were developed using enhanced chemiluminescence reagent (GE Healthcare) and imaged (LI-COR C-DiGit Chemiluminescence Western Blot Scanner). The band intensities were determined using ImageJ Software (NIH). Blots were stripped using stripping buffer from thermo scientific according to manufacturer protocols and reprobed using with an antiGAPDH as internal control to monitor the level of protein.
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