Western blot was performed using the protocol as previously described (19 (link)). The primary antibodies were anti-Phosph-4EBP1 (Thr37/46), anti-4EBP1 (Cell Signaling) and anti-actin (Santa Cruz Technology, CA). Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the optical density was analyzed using a Biospetrum 500 imaging system (UVP, LLC).
Biospetrum 500 imaging system
Manufactured by Analytik Jena
The Biospetrum 500 imaging system is a compact and versatile instrument designed for various imaging applications. It features a high-resolution camera, adjustable lighting, and a motorized stage for sample positioning. The system is capable of capturing detailed images and documentation of a wide range of samples, including cells, gels, and other biological and chemical specimens.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Antibody anti sesn1 ab134091, by Abcam (2 mentions)
Anti nf κb p50 sc 8414, by Santa Cruz Biotechnology (2 mentions)
Phospho ikkα β ser176 180 antibody 2694, by Cell Signaling Technology (2 mentions)
Ikkβ antibody 8943, by Cell Signaling Technology (2 mentions)
β actin, by Analytik Jena (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Nuclear extraction kit, by Abcam (2 mentions)
Anti β actin sc 47778, by Santa Cruz Biotechnology (2 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Rabbit monoclonal anti mtor, by Cell Signaling Technology (1 mentions)
Anti phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using biospetrum 500 imaging system
1
Phospho-4EBP1 Western Blot Protocol
2
Antibody-Based Protein Analysis
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The following primary antibodies were used: rabbit monoclonal anti-mTOR and anti-phospho-mTOR (Ser2448) were purchased from Cell Signaling Technology. Mouse monoclonal anti-β-actin was purchased from Santa Cruz Biotechnology. Rabbit monoclonal anti-Sestrin2 was purchased from Abcam. Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and were visualized and the optical density of the identified protein bands on membranes was analyzed using a Biospetrum 500 imaging system (UVP, LLC).
3
Protein Extraction and Quantification Protocol
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Total cellular proteins were extracted by incubating cells in RIPA buffer (Thermo Fisher) on ice for 30min. The nuclear proteins were isolated using the Nuclear Extraction Kit (Abcam). Antibody anti-SESN1 (ab134091) was purchased from Abcam. Anti-β-actin (sc-47778) and anti-NF-κB p50 (sc-8414) were bought from Santa Cruz Biotechnology. IκBα antibody (4814), NF-κB p65 antibody (8242), phospho-IKKα/ β (Ser176/180) antibody (2694), and IKKβ antibody (8943) were purchased from Cell Signaling Technology. The densitometry was done on a Biospetrum 500 imaging system (UVP, LLC) and quanti ed with ImageJ. The target proteins were normalized to β-actin. The normalized target proteins in treatment groups were then divided by normalized target proteins in control groups. To quantify IKKβ phosphorylation, the phosphorylated IKKβ was normalized to total IKKβ, and then normalized phosphorylated IKKβ in treatment groups were then divided by normalized phosphorylated IKKβ in control groups.
4
Protein Extraction and Quantification Protocol
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Total cellular proteins were extracted by incubating cells in RIPA buffer (Thermo Fisher) on ice for 30min. The nuclear proteins were isolated using the Nuclear Extraction Kit (Abcam). Antibody anti-SESN1 (ab134091) was purchased from Abcam. Anti-β-actin (sc-47778) and anti-NF-κB p50 (sc-8414) were bought from Santa Cruz Biotechnology. IκBα antibody (4814), NF-κB p65 antibody (8242), phospho-IKKα/ β (Ser176/180) antibody (2694), and IKKβ antibody (8943) were purchased from Cell Signaling Technology. The densitometry was done on a Biospetrum 500 imaging system (UVP, LLC) and quanti ed with ImageJ. The target proteins were normalized to β-actin. The normalized target proteins in treatment groups were then divided by normalized target proteins in control groups. To quantify IKKβ phosphorylation, the phosphorylated IKKβ was normalized to total IKKβ, and then normalized phosphorylated IKKβ in treatment groups were then divided by normalized phosphorylated IKKβ in control groups.
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