Western blotting was performed as described earlier [24] (link), [25] (link), [26] (link) with modifications. Briefly, cells were scraped in lysis buffer, transferred to microfuge tubes and spun into pellet. The supernatant was collected and analyzed for protein concentration via the Bradford method (Bio-Rad). SDS sample buffer was added to 30–50 µg total protein and the sample was boiled for 5 min. Denatured samples were electrophoresed on NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen) and proteins transferred onto a nitrocellulose membrane (Bio-Rad) using the Thermo-Pierce Fast Semi-Dry Blotter. The membrane was then washed for 15 min in TBS plus Tween 20 (TBST) and blocked for 1 h in TBST containing BSA. Next, membranes were incubated overnight at 4°C under shaking conditions with primary antibody. The next day, membranes were washed in TBST for 1 h, incubated with secondary antibody (Li-Cor Biosciences) for 1 h at room temperature, washed for one more hour and visualized under the Odyssey Infrared Imaging System (Li-COR, Lincoln, NE).
Anitrocellulose membrane
Manufactured by Bio-Rad
Sourced in United States
Nitrocellulose membrane is a porous material made from cellulose that is commonly used in various laboratory techniques, such as Western blotting, dot blotting, and other protein-based assays. It provides a surface for the immobilization and detection of proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Bradford method, by Bio-Rad (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Odyssey infrared fluorescent imaging system, by LI COR (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Proper fluorescent secondary antibodies, by LI COR (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
Camk2a, by Cell Signaling Technology (1 mentions)
Uchl1, by Cell Signaling Technology (1 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Sheep anti mouse antibody conjugated to horseradish peroxidase hrp, by GE Healthcare (1 mentions)
Sv5 pk1, by Abcam (1 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
Adipor2, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit antibody, by Bioworld Technology (1 mentions)
5 protocols using anitrocellulose membrane
1
Western Blot with Modifications
2
Quantitative Western Blot Analysis of PSD Proteins
3
Western Blot Protein Detection
4
Yeast Protein Extraction and Western Blot
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Cells were grown at 30ºC in YPA medium (1% yeast extract, 2% bactopeptone, 0.002% adenine) supplemented with 2% dextrose (Sherman 1991) to an OD 660 of 1.0 to 1.5. For each sample, 1.0 OD of cells was collected, and protein lysate was prepared by pulverizing cells with glass beads in sodium dodecyl sulfate (SDS) buffer. Standard methods for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used. A nitrocellulose membrane (Bio-Rad) was used for western transfer with standard methods. Immunoblots were incubated with a 1:5,000 dilution of mouse monoclonal antibody to the V5 epitope SV5-Pk1; Abcam) or a 1:10,000 dilution of mouse monoclonal antibody to Pgk1 (Invitrogen), followed by a 1:10,000 dilution of sheep anti-mouse antibody conjugated to horseradish peroxidase (HRP) (GE Biosciences). Antibody signals were detected with Super Signal West Dura Chemiluminescent Substrate (Thermo Scientific).
5
Protein Expression Analysis via Western Blot
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Proteins were extracted inlysis bufferaccording to the manufacturer's protocol. Lysates were separated by SDS-PAGEand transferred to anitrocellulose membrane (Bio-Rad, USA).The membranes were incubated in blocking buffer.Themembranes were incubated with the AdipoR2 (Abcam, USA), AMPK, phosphorylated (Thr172) AMPK (p-AMPK), mTOR, phosphorylated (Ser2448) mTOR (p-mTOR),70-kDa ribosomal protein S6 kinase(S6K), phosphorylated(Thr421/Ser424) p70S6 kinase (pS6K), and S6 ribosomal protein (S6P), phosphorylated(Ser240/244) S6 ribosomal protein (pS6P)and GAPDH (CST, USA) primary antibodyat 4 °Covernight, respectively. Immunoreactivity was visualizedwith horseradish peroxidase-conjugated goat anti-rabbit antibody (Bioworld, USA). Protein bands were detected and imaged with ChemiDocXRS + gel imaging system (Bio-Rad, USA)and analyzed bydensitometric quanti cation using Image J software.
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