Cell lysates for immunoblotting were directly lysed in Laemmli buffer (Roth) and sonicated for 30 min on ice or in RIPA buffer (SantaCruz) supplemented with complete protease-inhibitor cocktail (Roche). Protein quantification was performed with the Pierce 660 nm Protein Assay (Pierce). Alternatively, samples were prepared in RIPA buffer and mixed in equal amounts with Laemmli buffer (Roth) before denaturation at 98°C for 5 min. Protein samples were separated via SDS-PAGE, blotted onto nitrocellulose membranes, blocked with 5% BSA and incubated with the primary antibodies (see Key Resources Table ). Detection was carried out with goat anti-mouse or anti-rabbit horse radish peroxidase (HRP) conjugated secondary antibodies (BioRad), visualized with the enhanced chemiluminescent SuperSignal reagent (Pierce) and detected using a digital ChemiDoc imaging system (BioRad).
Laemmli buffer
Manufactured by Carl Roth
Laemmli buffer is a common sample buffer used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the separation of proteins based on their molecular weight. It contains the anionic detergent SDS, which denatures proteins by breaking down their secondary and non-disulfide-linked tertiary structures, allowing them to migrate through the gel based on their size.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Rabbit anti pgc 1α, by Abcam (1 mentions)
Rabbit anti tfam, by Abcam (1 mentions)
Goat anti rabbit horseradish peroxidase hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
2 protocols using laemmli buffer
1
Western Blot Sample Preparation
2
Immunoblotting of T Cell Metabolism
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Cell lysates were prepared in RIPA buffer supplemented with a complete protease-inhibitor cocktail (Roche) and mixed in equal amounts with Laemmli buffer (Roth) before denaturation at 98 °C for 5 min. Protein quantification was performed with the Pierce 660 nm Protein Assay (Pierce). Protein samples were separated via SDS-PAGE, blotted onto nitrocellulose membranes, blocked with 5% BSA, and incubated with monoclonal rabbit anti-HIF-1α (Cell Signaling Technology, clone D1S7W). Detection was carried out with a goat anti-rabbit horse radish peroxidase (HRP) conjugated secondary antibody (BioRad), visualized with the enhanced chemiluminescent SuperSignal reagent (Pierce). For the detection of PGC-1α, TFAM, and elector transport chain (ETC) complex expression, Tpex and Tex cells were FACS sorted from LCMVCL13 infected mice 30 d.p.i. and 5x105 T cells were directly lysed in RIPA buffer. Detection of PGC-1α, TFAM, and ETC complexes was carried out using rabbit anti-PGC-1α (Abcam), rabbit-anti-TFAM (Abcam), and an OxPhos rodent antibody cocktail (Thermo) with goat anti-mouse or rabbit HRP-conjugated secondary antibodies (both BioRad).
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